Compounds as peptidic glp1/glucagon/gip receptor agonists

ABSTRACT

The present invention relates to trigonal GLP-1/glucagon/GIP receptor agonists and their medical use, for example in the treatment of disorders of the metabolic syndrome, including diabetes and obesity, as well as for reduction of excess food intake.

RELATED APPLICATION

This application claims priority to European Patent Application No.16306605.3, filed Dec. 2, 2016, the entire disclosure of which is herebyincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to new compounds which are trigonalGLP-1/glucagon/GIP receptor agonists and their medical use, for examplein the treatment of disorders of the metabolic syndrome, includingdiabetes and obesity, as well as for reduction of excess food intake.The compounds of the invention are structurally derived from exendin-4and show high solubility and stability under acidic conditions in thepresence of antimicrobial preservatives like m-cresol or phenol whichmakes them especially suited for combinations with other antidiabeticcompounds.

BACKGROUND OF THE INVENTION

Bhat et al (Diabetologia 2013, 56, 1417-1424), Bhat et al. (BiochemPharmacol. 2013, 85, 1655-62), Gault et al. (J Biol Chem. 2013, 288,35581-91) as well as Finan et al. (Nat Med. 2015, 21, 27-36) describedtrigonal agonists of the glucagon-like peptide-1 (GLP-1), the glucagonand the glucose-dependent insulinotropic polypeptide (GIP) receptors,e.g. by combining the actions of GLP-1, glucagon and GIP in onemolecule, which leads to a therapeutic principle with anti-diabeticaction and a pronounced weight lowering effect superior to pure GLP-1agonists, among others due to glucagon-receptor mediated increasedsatiety and energy expenditure as well as GIP receptor mediatedincreased insulin secretion.

The amino acid sequence of GLP-1(7-36)-amide is shown as SEQ ID NO: 1.

HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH₂

Liraglutide is a marketed chemically modified GLP-1 analog in which,among other modifications, a fatty acid is linked to a lysine inposition 20 leading to a prolonged duration of action (Drucker et al,Nat. Rev. Drug Disc. 2010, 9, 267-268; Buse et al., Lancet 2009, 374,39-47).

The amino acid sequence of Liraglutide is shown as SEQ ID NO: 2.

HAEGTFTSDVSSYLEGQAAK((S)-4-Carboxy-4-hexadecanoylamino-butyryl)EFIAWLVRGRG-OH

Glucagon is a 29-amino acid peptide which is released into thebloodstream when circulating glucose is low. Glucagon's amino acidsequence is shown as SEQ ID NO: 3.

HSQGTFTSDYSKYLDSRRAQDFVQWLMNT-OH

During hypoglycemia, when blood glucose levels drop below normal,glucagon signals the liver to break down glycogen and release glucose,causing an increase of blood glucose levels to reach a normal level.Recent publications suggest that glucagon has in addition beneficialeffects on reduction of body fat mass, reduction of food intake, andincrease of energy expenditure (Heppner et al., Physiology & Behavior2010, 100, 545-548).

GIP (glucose-dependent insulinotropic polypeptide) is a 42 amino acidpeptide that is released from intestinal K-cells following food intake.GIP and GLP-1 are the two gut enteroendocrine cell-derived hormonesaccounting for the incretin effect, which accounts for over 70% of theinsulin response to an oral glucose challenge (Baggio et al.Gastroenterology 2007, 132, 2131-2157).

GIP's amino acid sequence is shown as SEQ ID NO: 5.

YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ-OH

Peptides which are based on the structures of GLP-1 or glucagon, andbind and activate the GLP-1, the glucagon and the GIP receptor have beendescribed in patent applications WO 2010/011439, WO 2010/148089, WO2012/088116, WO 2013/192129, WO 2013/192130, WO 2014/049610 and WO2015/067716. Further trispecific agonists based on exendin-4 have beendescribed in WO 2014/096145, WO 2015/086731, WO 2015/086732, WO2015/086733, WO2015/155141, and PCT/EP2016/063332. The compoundsdescribed therein have been shown to lead to improved glycemic control,possible islet and beta-cell preservation and enhanced body weight loss.

Peptides which bind and activate both the GIP and the GLP-1 receptordesigned as analogues of exendin-4 and substituted with a fatty acidside chain are described in patent applications WO 2014/096145 A1, WO2014/096150 A1, WO 2014/096149 A1, and WO 2014/096148 A1.

Exendin-4 is a 39 amino acid peptide which is produced by the salivaryglands of the Gila monster (Heloderma suspectum. Exendin-4 is anactivator of the GLP-1 receptor, whereas it shows low activation of theGIP receptor and does not activate the glucagon receptor (see Table 1).

TABLE 1 Potencies of exendin-4 at human GLP-1, GIP and Glucagonreceptors (indicated in pM) at increasing concentrations and measuringthe formed cAMP as described in Methods. SEQ ID EC50 hGLP- EC50 hGIPEC50 hGlucagon NO: Peptide 1R [pM] R [pM] R [pM] 4 exendin-4 0.4 12500.0>10000000

The amino acid sequence of exendin-4 is shown as SEQ ID NO: 4.

HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2

Exendin-4 shares many of the glucoregulatory actions observed withGLP-1. Clinical and nonclinical studies have shown that exendin-4 hasseveral beneficial antidiabetic properties including a glucose dependentenhancement in insulin synthesis and secretion, glucose dependentsuppression of glucagon secretion, slowing down gastric emptying,reduction of food intake and body weight, and an increase in beta-cellmass and markers of beta cell function.

These effects may be beneficial not only for diabetics but also forpatients suffering from obesity. Patients with obesity have a higherrisk of getting diabetes, hypertension, hyperlipidemia, cardiovascularand musculoskeletal diseases.

Relative to GLP-1, exendin-4 is resistant to cleavage by dipeptidylpeptidase-4 (DPP4) resulting in a longer half-life and duration ofaction in vivo (Eng J., Diabetes, 1996, 45 (Suppl 2):152A (abstract554)).

Exendin-4 was also shown to be much more stable towards degradation byneutral endopeptidase (NEP), when compared to GLP-1, glucagon oroxyntomodulin (Druce et al., Endocrinology, 2009, 150(4), 1712-1722).Nevertheless, exendin-4 is chemically labile due to methionine oxidationin position 14 (Hargrove et al., Regul. Pept., 2007, 141, 113-119) aswell as deamidation and isomerization of asparagine in position 28 (WO2004/035623).

Bloom et al. (WO 2006/134340) disclose that peptides which bind andactivate both the glucagon and the GLP-1 receptor can be constructed ashybrid molecules from glucagon and exendin-4, where the N-terminal part(e.g. residues 1-14 or 1-24) originates from glucagon and the C-terminalpart (e.g. residues 15-39 or 25-39) originates from exendin-4. Suchpeptides comprise glucagon's amino acid motif YSKY in position 10-13.Krstenansky et al (Biochemistry, 1986, 25, 3833-3839) show theimportance of these residues 10-13 of glucagon for its receptorinteractions and activation of adenylate cyclase.

Compared to GLP-1, glucagon and oxyntomodulin, exendin-4 has beneficialphysicochemical properties, such as solubility and stability in solutionand under physiological conditions (including enzymatic stabilitytowards degradation by enzymes, such as DPP4 or NEP), which results in alonger duration of action in vivo.

Nevertheless, also exendin-4 has been shown to be chemically labile dueto methionine oxidation in position 14 as well as deamidation andisomerization of asparagine in position 28. Stability might be furtherimproved by substitution of methionine at position 14 and the avoidanceof sequences that are known to be prone to degradation via aspartimideformation, especially Asp-Gly or Asn-Gly at positions 28 and 29.

DESCRIPTION OF THE INVENTION

In the compounds of the present invention several of the underlyingresidues are different from glucagon and the peptides described in WO2006/134340. In particular residues Tyr10 and Tyr13, which are known tocontribute to the fibrillation of glucagon (J S Pedersen et al.,Biochemistry, 2006, 45, 14503-14512) are replaced by Leu. Thisreplacement, especially in combination with isoleucine in position 23and glutamate in position 24, leads to exendin-4 derivatives withpotentially improved biophysical properties as solubility or aggregationbehaviour in solution. The non-conservative replacement of an aromaticamino acid with an aliphatic amino acid in position 13 of an exendin-4analogue leads to peptides with high activity on both the glucagon andthe GIP receptor, keeping their activity on the GLP-1 receptor.

Native exendin-4 is a pure GLP-1 receptor agonist without activity onthe glucagon receptor and low activity on the GIP receptor. Thecompounds of the invention are based on the structure of nativeexendin-4 but differing at fourteen or more positions as compared to SEQID NO: 4 wherein the differences contribute to the enhancement of theagonistic activity at the glucagon receptor and the GIP receptor. Amongother substitutions—methionine at position 14 is replaced by an aminoacid carrying an —NH₂ group in the side-chain, which is furthersubstituted by a lipophilic residue (e.g. a fatty acid combined with alinker). Further the replacement of the exendin-4 amino acids atpositions 13, 19, 20, 32, 34, 35 and 39 with a Leu in position 13, a Glnin position 19, an Aib or Lys amino acid in position 20, an Aib inposition 34, Pro at position 32 and Lys at position 35 and 39 leads tohigh activity on both the glucagon as well as the GIP receptor whilekeeping the high activity on the GLP-1 receptor. These peptides alsoshow high chemical stability, solubility and physical stability atacidic pH values, such as pH4.5.

Compounds of the invention have the formula I:

I H₂N-His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Leu-X14-Glu-X16-Gln-Arg-Gln-Aib-Glu-Phe-Ile-Glu-Trp-Leu-Lys-Ala-X29-Gly-X31-Pro-Ser-Aib-Lys- Pro-Pro-Pro-Lys-R¹wherein

-   -   X14 represents an amino acid residue with a functionalized —NH₂        side chain group, selected from the group consisting of Lys,        Orn, Dab, or Dap, wherein the —NH₂ side chain group is        functionalized by —Z—C(O)—R⁵, wherein        -   Z represents a linker in all stereoisomeric forms and        -   R⁵ is a moiety comprising up to 50 carbon atoms and            heteroatoms selected from N and O,    -   X16 represents an amino acid residue selected from Lys and Glu,    -   X29 represents an amino acid residue selected from D-Ala and        Gly,    -   X31 represents an amino acid residue selected from His and Pro,    -   R¹ is NH₂ or OH,        -   or a salt or solvate thereof.

The compounds of the invention are GLP-1, glucagon and GIP receptoragonists as determined by the observation that they are capable ofstimulating intracellular cAMP formation in the assay system describedin Methods.

According to another embodiment the compounds of the invention,particularly with a lysine at position 14 which is further substitutedwith a lipophilic residue, exhibit at least a relative activity of 0.1%(i.e. EC₅₀<700 pM), more preferably of 1% (i.e. EC₅₀<70 pM), morepreferably of 5% (i.e. EC₅₀<14 pM) and even more preferably of 10% (i.e.EC₅₀<7 pM) compared to that of GLP-1(7-36)amide at the GLP-1 receptor.Furthermore, the compounds of the invention exhibit at least a relativeactivity of 0.1% (i.e. EC₅₀<500 pM), more preferably of 0.25% (i.e.EC₅₀<200 pM), more preferably of 0,4% (<125 pM) and even more preferablyof 1% (i.e. EC₅₀<50 pM) compared to that of natural glucagon at theglucagon receptor. Furthermore, the compounds of the invention exhibitat least a relative activity of 0.1% (i.e. EC₅₀<400 pM), more preferablyof 0.4% (i.e. EC₅₀<100 pM), more preferably of 1% (<40 pM) and even morepreferably of 2% (i.e. EC₅₀<20 pM) compared to that of natural GIP atthe GIP receptor.

The term “activity” as used herein preferably refers to the capabilityof a compound to activate the human GLP-1 receptor, the human glucagonreceptor and the human GIP receptor. More preferably the term “activity”as used herein refers to the capability of a compound to stimulateintracellular cAMP formation. The term “relative activity” as usedherein is understood to refer to the capability of a compound toactivate a receptor in a certain ratio as compared to another receptoragonist or as compared to another receptor. The activation of thereceptors by the agonists (e.g. by measuring the cAMP level) isdetermined as described herein, e.g. as described in the examples.

The compounds of the invention preferably have an EC₅₀ for hGLP-1receptor of 450 pM or less, preferably of 200 pM or less, morepreferably of 100 pM or less, more preferably of 50 pM or less, morepreferably of 25 pM or less, more preferably of 10 pM or less, morepreferably of 8 pM or less, and more preferably of 5 pM or less and anEC₅₀ for hGlucagon receptor of 450 pM or less, preferably of 200 pM orless, more preferably of 100 pM or less, more preferably of 50 pM orless, and an EC₅₀ for hGIP receptor of 450 pM or less, preferably of 200pM or less, more preferably of 100 pM or less, more preferably of 50 pMor less, more preferably of 25 pM or less. The EC₅₀ for the hGLP-1receptor, the hGlucagon receptor and the hGIP receptor may be determinedas described in the Methods herein and as used to generate the resultsdescribed in the examples.

Compounds of the formula I particularly those with a lysine at position14 which is further substituted with a lipophilic residue, showedincreased glucagon receptor activation compared to derivatives havingthe original methionine (from exendin-4) or leucine at position 14 (seealso WO2014/056872). Furthermore, oxidation (in vitro or in vivo) ofmethionine is not possible anymore.

The compounds of formula I do not only show high activity on theglucagon receptor but as well on the GIP receptor. The additional highactivity on the GIP receptor is intended for enhanced efficacy on bloodglucose control compared to pure GLP-1 receptor agonism and to reducethe probability of GLP-1 related side effects like gastrointestinaldistress as the contribution of the GLP-1 part can be reduced. Theadditional GIP receptor activity is also intended to counterbalance apotential glucose increase by glucagon receptor activation thereforeallowing higher glucagon receptor activity as observed in compounds offormula I (Finan et al. Nat Med. 2015, 21, 27-36).

In one embodiment the compounds of the invention have a high solubilityat acidic and/or physiological pH values in the presence of anantimicrobial preservative like phenol or m-cresol, e.g., at an acidityrange from pH 4 to 5, especially pH 4.5 and/or a more physiologicalrange from pH 6 to 8, especially at pH 7.4 at 25° C. or 40° C., inanother embodiment at least 1 mg/ml and in a particular embodiment atleast 5 mg/ml.

Furthermore, the compounds of the invention preferably have a highstability when stored in solution in the presence of an antimicrobialpreservative like phenol or m-cresol. Preferred assay conditions fordetermining the stability is storage for 28 days at 25° C. or 40° C. insolution at an acidity range from pH 4 to 5, especially pH 4.5. Thestability of peptide is determined by chromatographic analyses asdescribed in the Methods. Preferably, after 28 days at 40° C. insolution at pH 4.5 the purity loss is no more than 20%, more preferablyno more than 15%, more preferably no more than 12% and even morepreferably no more than 10%.

In one embodiment the compounds of the invention show a hydrodynamicradius R_(h) of 5 nm or less at concentrations of 1 mg/ml in thepresence of an antimicrobial preservative like phenol or m-cresol, e.g.,at an acidity range from pH 4 to 5 at 25° C., especially pH 4.5 at 25°C. as assayed by dynamic light scattering as described in Methods.

In one embodiment the compounds of the invention do not show an increasein fluorescence intensity with Thioflavin T as fluorescence probe atconcentrations of 3 mg/ml in the presence of an antimicrobialpreservative like phenol or m-cresol, e.g., at an acidity range from pH4 to 5, especially pH 4.5 at 37° C. over 5 hours, more preferably over10 hours, more preferably over 20 hours, more preferably over 30 hours,more preferably over 40 hours and even more preferably over 45 hours asassayed by the Tht assay as described in Methods.

In one embodiment the compounds of this invention are more resistant tocleavage by neutral endopeptidase (NEP) and dipeptidyl peptidase-4(DPP4), resulting in a longer half-life and duration of action in vivo,when compared with native GLP-1 and glucagon.

In one embodiment the compounds of the present invention comprise apeptide moiety which is a linear sequence of 39 amino carboxylic acids,particularly α-amino carboxylic acids linked by peptide, i.e.carboxamide bonds.

In one embodiment, R¹ is NH₂.

Specific preferred examples for —Z—C(O)—R⁵ groups are listed in thefollowing Table 2, which are selected from

-   (S)-4-Carboxy-4-hexadecanoylamino-butyryl-,    (S)-4-Carboxy-4-octadecanoylamino-butyryl-,    (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl,    (4S)-Carboxy-[2-(2-{2-[(4R)-5-carboxy-4-hexadecanoylamino-pentanoylamino]-ethoxy}-ethoxy)-acetylamino]-butyryl,    (4S)-Carboxy-[2-(2-{2-[(4R)-5-carboxy-4-hexadecanoylamino-pentanoylamino]-ethoxy}-ethoxy)-acetylamino]-butyryl,    (2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-hexadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl,    (2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-octadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl,    [2-(2-{2-[2-(2-{2-[2-(2-Octadecanoylamino-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl-.

Further preferred are stereoisomers, particularly enantiomers of thesegroups, either S- or R-enantiomers. The term “R” in Table 2 is intendedto mean the attachment site of —Z—C(O)—R⁵ at the peptide back bone, forexample the epsilon-amino group of Lys.

TABLE 2 Structure/IUPAC name

gGlu- Stea (S)-4-Carboxy-4-octadecanoylamino-butyryl-

gGlu- Palm (S)-4-Carboxy-4-hexadecanoylamino-butyryl-

gGlu- gGlu- Palm(S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-

gGlu- gGlu- AEEAc- Palm [[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-(hexadecanoylamino)ethoxy]ethoxy]acetyl]amino]butyrylamino]-butyryl-

gGlu- AEEAc- gAAA- Palm(4S)-Carboxy-[2-(2-{2-[(4R)-5-carboxy-4-hexadecanoylamino-pentanoylamino]-ethoxy}-ethoxy)acetylamino]butyryl

AEEAc- AEEAc- gGlu- Palm(2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-hexadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl

AEEAc- AEEAc- gGlu- Stea(2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-octadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl

AEEAc- AEEAc- AEEAc- Stea[2-(2-{2-[2-(2-{2-[2-(2-Octadecanoylamino-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl-

A further embodiment relates to compounds of formula I, wherein

-   -   X14 represents Lys wherein the —NH₂ side chain group is        functionalized with a group —Z—C(O)R⁵, wherein    -   Z represents a group selected from gGlu, gGlu-gGlu,        gGlu-AEEAc-gAAA-, gGlu-gGlu-AEEAc, AEEAc-AEEAc-gGlu and        AEEAc-AEEAc-AEEAc; and    -   R⁵ represents a group selected from pentadecanyl or        heptadecanyl.

A further embodiment relates to compounds of formula I, wherein

-   -   X14 represents Lys wherein the —NH₂ side chain group is        functionalized with a group —Z—C(O)R⁵, wherein    -   Z represents a group selected from gGlu, gGlu-gGlu,        gGlu-AEEAc-gAAA- and gGlu-gGlu-AEEAc; and    -   R⁵ represents a group selected from pentadecanyl or        heptadecanyl.

A further embodiment relates to compounds of formula I, wherein

-   -   X14 represents Lys wherein the —NH₂ side chain group is        functionalized by (S)-4-Carboxy-4-hexadecanoylamino-butyryl-,        (S)-4-Carboxy-4-octadecanoylamino-butyryl-,        (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-,        (2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-hexadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl,        (2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-octadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl,        [2-(2-{2-[2-(2-{2-[2-(2-Octadecanoylamino-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl-,    -   R¹ represents NH₂,    -   or a salt or solvate thereof.

A further embodiment relates to compounds of formula I, wherein

-   -   X14 represents Lys wherein the —NH₂ side chain group is        functionalized by (S)-4-Carboxy-4-hexadecanoylamino-butyryl-,        (S)-4-Carboxy-4-octadecanoylamino-butyryl-,        (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-,        (2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-hexadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl,        (2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-octadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl,    -   R¹ represents NH₂,    -   or a salt or solvate thereof.

A further embodiment relates to compounds of formula I, wherein

-   -   X14 represents Lys wherein the —NH₂ side chain group is        functionalized by        (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-,    -   X16 represents an amino acid residue selected from Glu and Lys,    -   X29 represents an amino acid residue selected from D-Ala and        Gly,    -   X31 represents an amino acid residue selected from His and Pro,    -   R¹ represents NH₂,    -   or a salt or solvate thereof.

A further embodiment relates to compounds of formula I, wherein

-   -   X14 represents Lys wherein the —NH₂ side chain group is        functionalized by        (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-,    -   X16 represents an amino acid residue selected from Glu and Lys,    -   X29 represents Gly,    -   X31 represents an amino acid residue selected from His and Pro,    -   R¹ represents NH₂,    -   or a salt or solvate thereof.

A further embodiment relates to compounds of formula I, wherein

-   -   X14 represents Lys wherein the —NH₂ side chain group is        functionalized by        (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-,        (S)-4-Carboxy-4-octadecanoylamino-butyryl-,    -   X16 represents Glu,    -   X29 represents an amino acid residue selected from D-Ala and        Gly,    -   X31 represents His,    -   R¹ represents NH₂,    -   or a salt or solvate thereof.

A further embodiment relates to compounds of formula I, wherein

-   -   X14 represents Lys wherein the —NH₂ side chain group is        functionalized by        (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-,        (S)-4-Carboxy-4-octadecanoylamino-butyryl-,    -   X16 represents Glu,    -   X29 represents Gly,    -   X31 represents an amino acid residue selected from His and Pro,    -   R¹ represents NH₂,    -   or a salt or solvate thereof.

A further embodiment relates to compounds of formula I, wherein

-   -   X14 represents Lys wherein the —NH₂ side chain group is        functionalized by        (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-,        (S)-4-Carboxy-4-octadecanoylamino-butyryl-,    -   X16 represents Lys,    -   X29 represents an amino acid residue selected from D-Ala and        Gly,    -   X31 represents an amino acid residue selected from His and Pro,    -   R¹ represents NH₂,    -   or a salt or solvate thereof.

A further embodiment relates to compounds of formula I, wherein

-   -   X14 represents Lys wherein the —NH₂ side chain group is        functionalized by        (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-,        (S)-4-Carboxy-4-octadecanoylamino-butyryl-,    -   X16 represents an amino acid residue selected from Glu and Lys,    -   X29 represents an amino acid residue selected from D-Ala and        Gly,    -   X31 represents Pro,    -   R¹ represents NH₂,    -   or a salt or solvate thereof.

A further embodiment relates to compounds of formula I, wherein

-   -   X14 represents Lys wherein the —NH₂ side chain group is        functionalized by        (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-,        (S)-4-Carboxy-4-octadecanoylamino-butyryl-,    -   X16 represents Lys,    -   X29 represents an amino acid residue selected from D-Ala and        Gly,    -   X31 represents Pro,    -   R¹ represents NH₂,    -   or a salt or solvate thereof.

A still further embodiment relates to compounds of formula I, wherein

-   -   X14 represents Lys wherein the —NH₂ side chain group is        functionalized by        (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-,    -   X16 represents Lys,    -   X29 represents D-Ala,    -   X31 represents an amino acid residue selected from His and Pro,    -   R¹ represents NH₂,    -   or a salt or solvate thereof.

Specific examples of compounds of formula I are the compounds of SEQ IDNO: 6-23, as well as salts or solvates thereof.

Specific examples of compounds of formula I are the compounds of SEQ IDNO: 6, 9 and 11 as well as salts or solvates thereof.

A specific example of compounds of formula I is the compound of SEQ IDNO: 6, well as salts or solvates thereof.

A specific example of compounds of formula I is the compound of SEQ IDNO: 9, well as salts or solvates thereof.

A specific example of peptidic compounds of formula I is the compound ofSEQ ID NO: 11, well as salts or solvates thereof.

In a further aspect, the present invention relates to a compositioncomprising a compound of the invention in admixture with a carrier. Inpreferred embodiments, the composition is a pharmaceutically acceptablecomposition and the carrier is a pharmaceutically acceptable carrier.The compound of the invention may be in the form of a salt, e.g. apharmaceutically acceptable salt or a solvate, e.g. a hydrate. In stilla further aspect, the present invention relates to a composition for usein a method of medical treatment, particularly in human medicine.

The compounds of formula I are suitable for human treatment without anadditional therapeutically effective agent. In other embodiments,however, the compounds may be used together with at least one additionaltherapeutically active agent, as described in “combination therapy”.

The compounds of formula I are particularly suitable for the treatmentor prevention of diseases or disorders caused by, associated with and/oraccompanied by disturbances in carbohydrate and/or lipid metabolism,e.g. for the treatment or prevention of hyperglycemia, type 2 diabetes,impaired glucose tolerance, type 1 diabetes, obesity and metabolicsyndrome. Further, the compounds of the invention may be suitable forthe treatment or prevention of degenerative diseases, particularlyneurodegenerative diseases.

The compounds described find use, inter alia, in preventing weight gainor promoting weight loss. By “preventing” is meant inhibiting orreducing when compared to the absence of treatment, and is notnecessarily meant to imply complete cessation of a disorder.

The compounds of the invention may cause a decrease in food intakeand/or increase in energy expenditure, resulting in the observed effecton body weight. Independently of their effect on body weight, thecompounds of the invention may have a beneficial effect on circulatingcholesterol levels, being capable of improving lipid levels,particularly LDL, as well as HDL levels (e.g. increasing HDL/LDL ratio).

Thus, the compounds of the invention may be used for direct or indirecttherapy of any condition caused or characterised by excess body weight,such as the treatment and/or prevention of obesity, morbid obesity,obesity linked inflammation, obesity linked gallbladder disease, obesityinduced sleep apnea. They may also be used for treatment and preventionof the metabolic syndrome, diabetes, hypertension, atherogenicdyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease,or stroke. Their effects in these conditions may be as a result of orassociated with their effect on body weight, or may be independentthereof.

Medical uses include delaying or preventing disease progression in type2 diabetes, treating metabolic syndrome, treating obesity or preventingoverweight, for decreasing food intake, increase energy expenditure,reducing body weight, delaying the progression from impaired glucosetolerance (IGT) to type 2 diabetes; delaying the progression from type 2diabetes to insulin-requiring diabetes; and hepatic steatosis.

Definitions

The amino acid sequences of the present invention contain theconventional one letter and three letter codes for naturally occurringamino acids, as well as generally accepted three letter codes for otheramino acids, such as Aib (a-aminoisobutyric acid).

The term “native exendin-4” refers to native exendin-4 having thesequence HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂ (SEQ ID NO: 4).

The invention relates to peptidic compounds as defined in formula I.

The peptidic compounds of the present invention comprise a linearbackbone of amino carboxylic acids linked by peptide, i.e. carboxamidebonds. Preferably, the amino carboxylic acids are α-amino carboxylicacids and more preferably L-α-amino carboxylic acids, unless indicatedotherwise. The peptidic compounds preferably comprise a backbonesequence of 39 amino carboxylic acids.

Amino acids within the peptide moiety (formula I) can be considered tobe numbered consecutively from 1 to 39 in the conventional N-terminal toC-terminal direction. Reference to a “position” within peptidic moiety Ishould be constructed accordingly, as should reference to positionswithin native exendin-4 and other molecules, e.g., in exendin-4, His isat position 1, Gly at position 2, . . . , Met at position 14, . . . andSer at position 39.

An amino acid residue with an —NH₂ side chain group, e.g. Lys, Orn, Dabor Dap, is functionalized in that at least one H atom of the —NH₂ sidechain group is replaced by —Z—C(O)—R⁵, wherein R⁵ comprises a lipophilicmoiety, e.g. an acyclic linear or branched (C₃-C₃₀) saturated orunsaturated hydrocarbon group, which is unsubstituted or substitutede.g. by halogen (F, Cl, Br, J), —OH and/or CO₂H and Z comprises a linkerin all stereoisomeric forms, e.g. a linker comprising one or more, e.g.1 to 5, preferably 1, 2 or 3 amino acid linker groups selected from thegroup gamma-Glutamate (gGlu), gAAA and AEEAc. Preferred groups R⁵comprise a lipophilic moiety, e.g. an acyclic linear or branched(C₁₂-C₂₀) saturated or unsaturated hydrocarbon group, e.g. pentadecanyl,hexadecanyl or heptadecanyl, which is unsubstituted or substituted byCO₂H, more preferably pentadecanyl, or heptadecanyl. In one embodimentamino acid linker groups are selected from gGlu, gGlu-gGlu,gGlu-gGlu-AEEAc, gGlu-AEEAc-gAAA, AEEAc-AEEAc-gGlu andAEEAc-AEEAc-AEEAc. In another embodiment the amino acid linker group isgGlu. In another embodiment the amino acid linker group is gGlu-gGlu. Inanother embodiment the amino acid linker group is gGlu-gGlu-AEEAc. Inanother embodiment the amino acid linker group is gGlu-AEEAc-gAAA. Inanother embodiment the amino acid linker group is AEEAc-AEEAc-gGlu. Inanother embodiment the amino acid linker group is AEEAc-AEEAc-AEEAc.

In a further aspect, the present invention relates to a compositioncomprising a compound of the invention as described herein, or a salt orsolvate thereof, in admixture with a carrier.

The invention also relates to the use of a compound of the presentinvention for use as a medicament, particularly for the treatment of acondition as described in the specification.

The invention also relates to a composition wherein the composition is apharmaceutically acceptable composition, and the carrier is apharmaceutically acceptable carrier.

Peptide Synthesis

The skilled person is aware of a variety of different methods to preparepeptides. These methods include but are not limited to syntheticapproaches and recombinant gene expression. Thus, one way of preparingthese peptides is the synthesis in solution or on a solid support andsubsequent isolation and purification. A different way of preparing thepeptides is gene expression in a host cell in which a DNA sequenceencoding the peptide has been introduced. Alternatively, the geneexpression can be achieved without utilizing a cell system. The methodsdescribed above may also be combined in any way.

A preferred way to prepare the compounds of the present invention issolid phase synthesis on a suitable resin. Solid phase peptide synthesisis a well-established methodology (see for example: Stewart and Young,Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford, Ill.,1984; E. Atherton and R. C. Sheppard, Solid Phase Peptide Synthesis. APractical Approach, Oxford-IRL Press, New York, 1989). Solid phasesynthesis is initiated by attaching an N-terminally protected amino acidwith its carboxy terminus to an inert solid support carrying a cleavablelinker. This solid support can be any polymer that allows coupling ofthe initial amino acid, e.g. a trityl resin, a chlorotrityl resin, aWang resin or a Rink resin in which the linkage of the carboxy group (orcarboxamide for Rink resin) to the resin is sensitive to acid (when Fmocstrategy is used). The polymer support must be stable under theconditions used to deprotect the α-amino group during the peptidesynthesis.

After the N-terminally protected first amino acid has been coupled tothe solid support, the α-amino protecting group of this amino acid isremoved. The remaining protected amino acids are then coupled one afterthe other or with a preformed dipeptide, tripeptide or tetrapeptide inthe order represented by the peptide sequence using appropriate amidecoupling reagents, for example BOP, HBTU, HATU or DIC(N,N′-diisopropylcarbodiimide)/HOBt (1-hydroxybenzotriazole), whereinBOP, HBTU and HATU are used with tertiary amine bases. Alternatively,the liberated N-terminus can be functionalized with groups other thanamino acids, for example carboxylic acids, etc.

Usually, reactive side-chain groups of the amino acids are protectedwith suitable blocking groups. These protecting groups are removed afterthe desired peptides have been assembled. They are removed concomitantlywith the cleavage of the desired product from the resin under the sameconditions. Protecting groups and the procedures to introduce protectinggroups can be found in Protective Groups in Organic Synthesis, 3d ed.,Greene, T. W. and Wuts, P. G. M., Wiley & Sons (New York: 1999).

In some cases it might be desirable to have side-chain protecting groupsthat can selectively be removed while other side-chain protecting groupsremain intact. In this case the liberated functionality can beselectively functionalized. For example, a lysine may be protected withan ivDde ([1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl)protecting group (S. R. Chhabra et al., Tetrahedron Lett. 39, (1998),1603) which is labile to a very nucleophilic base, for example 4%hydrazine in DMF (dimethyl formamide). Thus, if the N-terminal aminogroup and all side-chain functionalities are protected with acid labileprotecting groups, the ivDde group can be selectively removed using 4%hydrazine in DMF and the corresponding free amino group can then befurther modified, e.g. by acylation. The lysine can alternatively becoupled to a protected amino acid and the amino group of this amino acidcan then be deprotected resulting in another free amino group which canbe acylated or attached to further amino acids. Alternatively, the sidechain (as described in table 2) can be introduced together with thelysine during peptide synthesis using a prefunctionalized buildingblock, e.g.(2S)-6-[[(4S)-5-tert-butoxy-4-[[(4S)-5-tert-butoxy-4-(hexadecanoylamino)-5-oxo-pentanoyl]amino]-5-oxo-pentanoyl]amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoicacid, as coupling partner.

Finally the peptide is cleaved from the resin. This can be achieved byusing King's cocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J.Peptide Protein Res. 36, 1990, 255-266). The raw material can then bepurified by chromatography, e.g. preparative RP-HPLC, if necessary.

Potency

As used herein, the term “potency” or “in vitro potency” is a measurefor the ability of a compound to activate the receptors for GLP-1,glucagon or GIP in a cell-based assay. Numerically, it is expressed asthe “EC50 value”, which is the effective concentration of a compoundthat induces a half maximal increase of response (e.g. formation ofintracellular cAMP) in a dose-response experiment.

Therapeutic Uses

Metabolic syndrome is a combination of medical disorders that, whenoccurring together, increase the risk of developing type 2 diabetes, aswell as atherosclerotic vascular disease, e.g. heart disease and stroke.Defining medical parameters for the metabolic syndrome include diabetesmellitus, impaired glucose tolerance, raised fasting glucose, insulinresistance, urinary albumin secretion, central obesity, hypertension,elevated triglycerides, elevated LDL cholesterol and reduced HDLcholesterol.

Obesity is a medical condition in which excess body fat has accumulatedto the extent that it may have an adverse effect on health and lifeexpectancy and due to its increasing prevalence in adults and childrenit has become one of the leading preventable causes of death in modernworld. It increases the likelihood of various other diseases, includingheart disease, type 2 diabetes, obstructive sleep apnea, certain typesof cancer, as well as osteoarthritis, and it is most commonly caused bya combination of excess food intake, reduced energy expenditure, as wellas genetic susceptibility.

Diabetes mellitus, often simply called diabetes, is a group of metabolicdiseases in which a person has high blood sugar levels, either becausethe body does not produce enough insulin, or because cells do notrespond to the insulin that is produced. The most common types ofdiabetes are: (1) type 1 diabetes, where the body fails to produceinsulin; (2) type 2 diabetes (T2DM), where the body fails to use insulinproperly, combined with an increase in insulin deficiency over time, and(3) gestational diabetes, where women develop diabetes due to theirpregnancy. All forms of diabetes increase the risk of long-termcomplications, which typically develop after many years. Most of theselong-term complications are based on damage to blood vessels and can bedivided into the two categories “macrovascular” disease, arising fromatherosclerosis of larger blood vessels and “microvascular” disease,arising from damage of small blood vessels. Examples for macrovasculardisease conditions are ischemic heart disease, myocardial infarction,stroke and peripheral vascular disease. Examples for microvasculardiseases are diabetic retinopathy, diabetic nephropathy, as well asdiabetic neuropathy.

The receptors for GLP-1 and GIP as well as glucagon are members of thefamily of 7-transmembrane-spanning, heterotrimeric G-protein coupledreceptors. They are structurally related to each other and share notonly a significant level of sequence identity, but have also similarmechanisms of ligand recognition and intracellular signaling pathways.

Similarly, the peptides GLP-1, GIP and glucagon share regions of highsequence identity/similarity. GLP-1 and glucagon are produced from acommon precursor, preproglucagon, which is differentially processed in atissue-specific manner to yield e.g. GLP-1 in intestinal endocrine cellsand glucagon in alpha cells of pancreatic islets. GIP is derived from alarger proGIP prohormone precursor and is synthesized and released fromK-cells located in the small intestine.

The peptidic incretin hormones GLP-1 and GIP are secreted by intestinalendocrine cells in response to food and account for up to 70% ofmeal-stimulated insulin secretion. Evidence suggests that GLP-1secretion is reduced in subjects with impaired glucose tolerance or type2 diabetes, whereas responsiveness to GLP-1 is still preserved in thesepatients. Thus, targeting of the GLP-1 receptor with suitable agonistsoffers an attractive approach for treatment of metabolic disorders,including diabetes. The receptor for GLP-1 is distributed widely, beingfound mainly in pancreatic islets, brain, heart, kidney and thegastrointestinal tract. In the pancreas, GLP-1 acts in a strictlyglucose-dependent manner by increasing secretion of insulin from betacells. This glucose-dependency shows that activation of GLP-1 receptorsis unlikely to cause hypoglycemia. Also the receptor for GIP is broadlyexpressed in peripheral tissues including pancreatic islets, adiposetissue, stomach, small intestine, heart, bone, lung, kidney, testis,adrenal cortex, pituitary, endothelial cells, trachea, spleen, thymus,thyroid and brain. Consistent with its biological function as incretinhormone, the pancreatic B-cell express the highest levels of thereceptor for GIP in humans.

There is some clinical evidence that the GIP-receptor mediated signalingcould be impaired in patients with T2DM but the impairment of GIP-actionis shown to be reversible and could be restored with improvement of thediabetic status. Of note, the stimulation of insulin secretion by bothincretin hormones, GIP and GLP-1, is strictly glucose-dependent ensuringa fail-safe mechanism associated with a low risk for hypoglycemia.

At the beta cell level, GLP-1 and GIP have been shown to promote glucosesensitivity, neogenesis, proliferation, transcription of proinsulin andhypertrophy, as well as anti-apoptosis. A peptide with both agonisticactivity for the GLP-1 and the GIP receptor could be anticipated to haveadditive or synergistic anti-diabetic benefit. Other relevant effects ofGLP-1 beyond the pancreas include delayed gastric emptying, increasedsatiety, decreased food intake, reduction of body weight, as well asneuroprotective and cardioprotective effects. In patients with type 2diabetes, such extrapancreatic effects could be particularly importantconsidering the high rates of comorbidities like obesity andcardiovascular disease. Further GIP actions in peripheral tissues beyondthe pancreas comprise increased bone formation and decreased boneresorption as well as neuroprotective effects which might be beneficialfor the treatment of osteoporosis and cognitive defects like Alzheimer'sdisease.

Glucagon is a 29 amino acid peptide hormone that is produced bypancreatic alpha cells and released into the bloodstream whencirculating glucose is low. An important physiological role of glucagonis to stimulate glucose output in the liver, which is a processproviding the major counterregulatory mechanism for insulin inmaintaining glucose homeostasis in vivo.

Glucagon receptors are however also expressed in extra-hepatic tissuessuch as kidney, heart, adipocytes, lymphoblasts, brain, retina, adrenalgland and gastrointestinal tract, suggesting a broader physiologicalrole beyond glucose homeostasis. Accordingly, recent studies havereported that glucagon has therapeutically positive effects on energymanagement, including stimulation of energy expenditure andthermogenesis, accompanied by reduction of food intake and body weightloss. Altogether, stimulation of glucagon receptors might be useful inthe treatment of obesity and the metabolic syndrome.

Oxyntomodulin is a peptide hormone consisting of glucagon with an eightamino acids encompassing C-terminal extension. Like GLP-1 and glucagon,it is pre-formed in preproglucagon and cleaved and secreted in atissue-specific manner by endocrinal cells of the small bowel.Oxyntomodulin is known to stimulate both, the receptors for GLP-1 andglucagon and is therefore the prototype of a dual agonist (see Pocai,Molecular Metabolism 2013; 3:241-51).

As GLP-1 and GIP are known for their anti-diabetic effects, GLP-1 andglucagon are both known for their food intake-suppressing effects andglucagon is also a mediator of additional energy expenditure, it isconceivable that a combination of the activities of the three hormonesin one molecule can yield a powerful medication for treatment of themetabolic syndrome and in particular its components diabetes andobesity.

Accordingly, the compounds of the invention may be used for treatment ofglucose intolerance, insulin resistance, pre-diabetes, increased fastingglucose (hyperglycemia), type 2 diabetes, hypertension, dyslipidemia,arteriosclerosis, coronary heart disease, peripheral artery disease,stroke or any combination of these individual disease components.

In addition, they may be used for control of appetite, feeding andcalorie intake, increase of energy expenditure, prevention of weightgain, promotion of weight loss, reduction of excess body weight andaltogether treatment of obesity, including morbid obesity.

The compounds of the invention are agonists for the receptors for GLP-1,GIP and for glucagon (e.g. “trigonal agonists”) and may providetherapeutic benefit to address a clinical need for targeting themetabolic syndrome by allowing simultaneous treatment of diabetes andobesity.

Further disease states and health conditions which could be treated withthe compounds of the invention are obesity-linked inflammation,obesity-linked gallbladder disease and obesity-induced sleep apnea.

Although all these conditions could be associated directly or indirectlywith obesity, the effects of the compounds of the invention may bemediated in whole or in part via an effect on body weight, orindependent thereof.

Further, diseases to be treated may be neurodegenerative diseases suchas Alzheimer's disease or Parkinson's disease, or other degenerativediseases as described above.

In one embodiment the compounds are useful in the treatment orprevention of hyperglycemia, type 2 diabetes and/or obesity.

The compounds of the invention may have the ability to reduce theintestinal passage, increase the gastric content and/or to reduce thefood intake of a patient. These activities of the compounds of theinvention can be assessed in animal models known to the skilled personand also described herein in the Methods.

The compounds of the invention have the ability to reduce blood glucoselevel, and/or to reduce HbA1c levels of a patient. These activities ofthe compounds of the invention can be assessed in animal models known tothe skilled person and also described herein in the Methods and inexamples.

The compounds of the invention may have the ability to reduce bodyweight of a patient. These activities of the compounds of the inventioncan be assessed in animal models known to the skilled person and alsodescribed herein in the Methods and in examples.

The compounds of the invention may be useful in the treatment orprevention of hepatosteatosis, preferably non-alcoholic liver-disease(NAFLD) and non-alcoholic steatohepatitis (NASH).

Pharmaceutical Compositions

The term “pharmaceutical composition” indicates a mixture containingingredients that are compatible when mixed and which may beadministered. A pharmaceutical composition may include one or moremedicinal drugs. Additionally, the pharmaceutical composition mayinclude carriers, buffers, acidifying agents, alkalizing agents,solvents, adjuvants, tonicity adjusters, emollients, expanders,preservatives, physical and chemical stabilizers e.g. surfactants,antioxidants and other components, whether these are considered activeor inactive ingredients. Guidance for the skilled in preparingpharmaceutical compositions may be found, for example, in Remington: TheScience and Practice of Pharmacy, (20th ed.) ed. A. R. Gennaro A. R.,2000, Lippencott Williams & Wilkins and in R. C. Rowe et al (Ed),Handbook of Pharmaceutical Excipients, PhP, May 2013 update.

The exendin-4 peptide derivatives of the present invention, or saltsthereof, are administered in conjunction with an acceptablepharmaceutical carrier, diluent, or excipient as part of apharmaceutical composition.

A “pharmaceutically acceptable carrier” is a carrier which isphysiologically acceptable (e.g. physiologically acceptable pH) whileretaining the therapeutic properties of the substance with which it isadministered. Standard acceptable pharmaceutical carriers and theirformulations are known to one skilled in the art and described, forexample, in Remington: The Science and Practice of Pharmacy, (20th ed.)ed. A. R. Gennaro A. R., 2000, Lippencott Williams & Wilkins and in R.C. Rowe et al (Ed), Handbook of Pharmaceutical excipients, PhP, May 2013update. One exemplary pharmaceutically acceptable carrier isphysiological saline solution.

In one embodiment carriers are selected from the group of buffers (e.g.citrate/citric acid, acetate/acetic acid), acidifying agents (e.g.hydrochloric acid), alkalizing agents (e.g. sodium hydroxide),preservatives (e.g. phenol, m-cresol), co-solvents (e.g. polyethyleneglycol 400), tonicity adjusters (e.g. mannitol, glycerol), stabilizers(e.g. surfactant, antioxidants, amino acids).

Concentrations used are in a range that is physiologically acceptable.

Acceptable pharmaceutical carriers or diluents include those used informulations suitable for oral, rectal, nasal or parenteral (includingsubcutaneous, intramuscular, intravenous, intradermal, and transdermal)administration. The compounds of the present invention will typically beadministered parenterally.

The term “pharmaceutically acceptable salt” means salts of the compoundsof the invention which are safe and effective for use in mammals.

The term “solvate” means complexes of the compounds of the invention orsalts thereof with solvent molecules, e.g. organic solvent moleculesand/or water.

In the pharmaceutical composition, the exendin-4 derivative can be inmonomeric or oligomeric form.

The term “therapeutically effective amount” of a compound refers to anontoxic but sufficient amount of the compound to provide the desiredeffect. The amount of a compound of the formula I necessary to achievethe desired biological effect depends on a number of factors, forexample the specific compound chosen, the intended use, the mode ofadministration and the clinical condition of the patient. An appropriate“effective” amount in any individual case may be determined by one ofordinary skill in the art using routine experimentation. For example the“therapeutically effective amount” of a compound of the formula I isabout 0.01 to 50 mg/dose, preferably 0.02 to 1 mg/dose.

Pharmaceutical compositions of the invention are those suitable forparenteral (for example subcutaneous, intramuscular, intradermal orintravenous), rectal, topical and peroral (for example sublingual)administration, although the most suitable mode of administrationdepends in each individual case on the nature and severity of thecondition to be treated and on the nature of the compound of formula Iused in each case. In one embodiment, application is parenteral, e.g.subcutaneous.

In case of parenteral application, it could be favorable for thecorresponding formulations to include at least one antimicrobialpreservative in order to inhibit the growth of microbes and bacteriabetween administrations. Preferred preservatives are benzylic alcohol orphenolic compounds like phenol or m-cresol. It has been described thatthese ingredients can induce aggregation for peptides and proteinsleading to lower solubility and stability in the formulation (see R. L.Bis et al., Int. J. Pharm. 472, 356-361, 2014; T. J. Kamerzell, Adv.Drug Deliv. Rev., 63, 1118-1159, 2011).

Suitable pharmaceutical compositions may be in the form of separateunits, for example capsules, tablets and powders in vials or ampoules,each of which contains a defined amount of the compound; as powders orgranules; as solution or suspension in an aqueous or non-aqueous liquid;or as an oil-in-water or water-in-oil emulsion. It may be provided insingle or multiple dose injectable form, for example in the form of apen. The compositions may, as already mentioned, be prepared by anysuitable pharmaceutical method which includes a step in which the activeingredient and the carrier (which may consist of one or more additionalingredients) are brought into contact.

In certain embodiments the pharmaceutical composition may be providedtogether with a device for application, for example together with asyringe, an injection pen or an autoinjector. Such devices may beprovided separate from a pharmaceutical composition or prefilled withthe pharmaceutical composition.

Administration Unit, Package, Pen Device and Administration

The compound(s) of the present invention can be prepared for use insuitable pharmaceutical compositions. The suitable pharmaceuticalcompositions may be in the form of one or more administration units.

The compositions may be prepared by any suitable pharmaceutical methodwhich includes a step in which the compound(s) of the present inventionand the carrier (which may consist of one or more additionalingredients) are brought into contact.

The administration units may be for example capsules, tablets, dragées,granules sachets, drops, solutions, suspensions, lyophylisates andpowders, each of which contains a defined amount of the compound(s) ofthe present invention.

Each of the above-mentioned administration units of the compound(s) ofthe invention or pharmaceutical composition of the invention(administration units) may be provided in a package for easy transportand storage. The administration units are packaged in standard single ormulti-dosage packaging, their form, material and shape depending on thetype of units prepared.

For example, tablets and other forms of solid administration units canbe packaged in single units, and the single packaged units can bepackaged in multi-pack containers. Liquid formulations can be packagedin single units, such as e.g. vials, cartridges, syringes/prefilledsyringes, infusion bags, collapsible plastic bags, infusion bottles,blow-filled seal bottles or infusion tubings or in single or multipledose injectable form, for example in the form of a pen device, pump orsyringe and the single packaged units can be packaged in multi-packcontainers. A single package may comprise only one or a plurality ofadministration units. The package may for example be made of paper,cardboard, paperboard, plastic, metal, combinations or laminates of oneor more of paper, plastics and metal, or glass. Exemplary embodimentsare blister packages containing e.g. tablets or capsules, which in turnmay be provided inside a cardboard box, aluminum barrier laminatesachets containing e.g. a powder, glass or plastic bottles containinge.g. tablets or a solution, or vials, cartridges, syringes, infusionbags, infusion bottles, infusion tubings or ampoules containing asolution or suspension.

In certain embodiments administration units may be provided togetherwith a device for application, for example together with a syringe, aninjection pen or an autoinjector. Such devices may be provided separatefrom a pharmaceutical composition or prefilled with the pharmaceuticalcomposition.

A “pen-type injection device”, often briefly referred to as “injectionpen”, is typically an injection device having an elongated shape thatresembles to a fountain pen for writing. Although such pens usually havea tubular cross-section, they could easily have a differentcross-section such as triangular, rectangular or square or any variationaround these geometries. Generally, pen-type injection devices comprisethree primary elements: a cartridge section that includes a cartridgeoften contained within a housing or holder; a needle assembly connectedto one end of the cartridge section; and a dosing section connected tothe other end of the cartridge section. The cartridge, often alsoreferred to as “ampoule”, typically includes a reservoir that is filledwith a medication, a movable rubber type bung or stopper located at oneend of the cartridge reservoir, and a top having a pierceable rubberseal located at the other, often necked-down, end. A crimped annularmetal band is typically used to hold the rubber seal in place. While thecartridge housing may be typically made of plastic, cartridge reservoirshave historically been made of glass.

Combination Therapy

The compounds of the present invention, trigonal agonists for the GLP-1,GIP and glucagon receptors, can be widely combined with otherpharmacologically active compounds, such as all drugs mentioned in theRote Liste 2016, e.g. with all weight-reducing agents or appetitesuppressants mentioned in the Rote Liste 2016, chapter 1, alllipid-lowering agents mentioned in the Rote Liste 2016, chapter 58, allantihypertensives and nephroprotectives, mentioned in the Rote Liste2016, or all diuretics mentioned in the Rote Liste 2015, chapter 36.

The active ingredient combinations can be used especially for asynergistic improvement in action. They can be applied either byseparate administration of the active ingredients to the patient or inthe form of combination products in which a plurality of activeingredients are present in one pharmaceutical preparation. When theactive ingredients are administered by separate administration of theactive ingredients, this can be done simultaneously or successively.

Other active substances which are suitable for such combinations includein particular those which for example potentiate the therapeutic effectof one or more active substances with respect to one of the indicationsmentioned and/or which allow the dosage of one or more active substancesto be reduced.

Therapeutic agents which are suitable for combinations include, forexample, antidiabetic agents such as:

Insulin and Insulin derivatives, for example: Glargine/Lantus®,270-330U/mL of insulin glargine (EP 2387989 A), 300 U/mL of insulinglargine (EP 2387989 A), Glulisin/Apidra®, Detemir/Levemir®,Lispro/Humalog®/Liprolog®, Degludec/DegludecPlus, Aspart, basal insulinand analogues (e.g. LY-2605541, LY2963016, NN1436), PEGylated insulinLispro, Humulin®, Linjeta, SuliXen®, NN1045, Insulin plus Symlin,PE0139, fast-acting and short-acting insulins (e.g. Linjeta, PH20,NN1218, HinsBet), (APC-002)hydrogel, oral, inhalable, transdermal andsublingual insulins (e.g. Exubera®, Nasulin®, Afrezza, Tregopil, TPM 02,Capsulin, Oral-lyn®, Cobalamin® oral insulin, ORMD-0801, NN1953, NN1954,NN1956, VIAtab, Oshadi oral insulin). Additionally included are alsothose insulin derivatives which are bonded to albumin or another proteinby a bifunctional linker.

GLP-1, GLP-1 analogues and GLP-1 receptor agonists, for example:Lixisenatide/AVE0010/ZP10/Lyxumia,Exenatide/Exendin-4/Byetta/Bydureon/ITCA 650/AC-2993,Liraglutide/Victoza, Semaglutide, Taspoglutide, Syncria/Albiglutide,Dulaglutide, rExendin-4, CJC-1134-PC, PB-1023, TTP-054,Efpeglenatide/HM-11260C, CM-3, GLP-1 Eligen, ORMD-0901, NN-9924,NN-9926, NN-9927, Nodexen, Viador-GLP-1, CVX-096, ZYOG-1, ZYD-1,GSK-2374697, DA-3091, MAR-701, MAR709, ZP-2929, ZP-3022, ZP-DI-70,TT-401, MK-8521, MED10382, BHM-034, HM12525A, MOD-6030, CAM-2036,DA-15864, ARI-2651, ARI-2255, LY3298176, NN1177, Exenatide-XTEN andGlucagon-XTEN, NN9030.

DPP-4 inhibitors, for example: Alogliptin/Nesina,Trajenta/Linagliptin/BI-1356/Ondero/Trajenta/Tradjenta/Trayenta/Tradzenta,Saxagliptin/Onglyza,Sitagliptin/Januvia/Xelevia/Tesave/Janumet/Velmetia,Galvus/Vildagliptin, Anagliptin, Gemigliptin, Teneligliptin,Melogliptin, Trelagliptin, DA-1229, Omarigliptin/MK-3102, KM-223,Evogliptin, ARI-2243, PBL-1427, Pinoxacin.

SGLT2 inhibitors, for example: Invokana/Canaglifozin,Forxiga/Dapagliflozin, Remoglifozin, Sergliflozin, Empagliflozin,Ipragliflozin, Tofogliflozin, Luseogliflozin, Sotagliflozin (LX-4211),Ertuglifozin/PF-04971729, RO-4998452, Bexagliflozin (EGT-0001442),KGA-3235/DSP-3235, LIK066, SBM-TFC-039, Henagliflozin (SHR3824),Janagliflozin, Tianagliflozin, AST1935, JRP493, HEC-44616

Biguanides (e.g. Metformin, Buformin, Phenformin), Thiazolidinediones(e.g. Pioglitazone, Rivoglitazone, Rosiglitazone, Troglitazone), dualPPAR agonists (e.g. Aleglitazar, Muraglitazar, Tesaglitazar),Sulfonylureas (e.g. Tolbutamide, Glibenclamide, Glimepiride/Amaryl,Glipizide), Meglitinides (e.g. Nateglinide, Repaglinide, Mitiglinide),Alpha-glucosidase inhibitors (e.g. Acarbose, Miglitol, Voglibose),Amylin and Amylin analogues (e.g. Pramlintide, Symlin).

GPR119 agonists (e.g. GSK-263A, PSN-821, MBX-2982, APD-597, ZYG-19,DS-8500), GPR40 agonists (e.g. Fasiglifam/TAK-875, TUG-424, P-1736,JTT-851, GW9508).

Other suitable combination partners are: Cycloset, inhibitors of11-beta-HSD (e.g. LY2523199, BMS770767, RG-4929, BMS816336, AZD-8329,HSD-016, BI-135585), activators of glucokinase (e.g. TTP-399, AMG-151,TAK-329, GKM-001), inhibitors of DGAT (e.g. LCQ-908), inhibitors ofprotein tyrosinephosphatase 1 (e.g. Trodusquemine), inhibitors ofglucose-6-phosphatase, inhibitors of fructose-1,6-bisphosphatase,inhibitors of glycogen phosphorylase, inhibitors of phosphoenol pyruvatecarboxykinase, inhibitors of glycogen synthase kinase, inhibitors ofpyruvate dehydrokinase, alpha 2-antagonists, CCR-2 antagonists, SGLT-1inhibitors (e.g. LX-2761).

One or more lipid lowering agents are also suitable as combinationpartners, such as for example: HMG-CoA-reductase inhibitors (e.g.Simvastatin, Atorvastatin), fibrates (e.g. Bezafibrate, Fenofibrate),nicotinic acid and the derivatives thereof (e.g. Niacin), PPAR-(alpha,gamma or alpha/gamma) agonists or modulators (e.g. Aleglitazar),PPAR-delta agonists, ACAT inhibitors (e.g. Avasimibe), cholesterolabsorption inhibitors (e.g. Ezetimibe), Bile acid-binding substances(e.g. Cholestyramine), ileal bile acid transport inhibitors, MTPinhibitors, or modulators of PCSK9.

HDL-raising compounds such as: CETP inhibitors (e.g. Torcetrapib,Anacetrapid, Dalcetrapid, Evacetrapid, JTT-302, DRL-17822, TA-8995) orABC1 regulators.

Other suitable combination partners are one or more active substancesfor the treatment of obesity, such as for example: Sibutramine,Tesofensine, Orlistat, antagonists of the cannabinoid-1 receptor, MCH-1receptor antagonists, MC4 receptor agonists, NPY5 or NPY2 antagonists(e.g. Velneperit), beta-3-agonists, leptin or leptin mimetics, agonistsof the 5HT2c receptor (e.g. Lorcaserin), or the combinations ofbupropione/naltrexone, bupropione/zonisamide, bupropione/phentermine orpramlintide/metreleptin.

Other suitable combination partners are:

Further gastrointestinal peptides such as Peptide YY 3-36 (PYY3-36) oranalogues thereof, pancreatic polypeptide (PP) or analogues thereof,glucagon receptor agonists or antagonists, GIP receptor agonists orantagonists, ghrelin antagonists or inverse agonists, Xenin andanalogues thereof.

Moreover, combinations with drugs for influencing high blood pressure,chronic heart failure or atherosclerosis, such as e.g.: Angiotensin IIreceptor antagonists (e.g. telmisartan, candesartan, valsartan,losartan, eprosartan, irbesartan, olmesartan, tasosartan, azilsartan),ACE inhibitors, ECE inhibitors, diuretics, beta-blockers, calciumantagonists, centrally acting hypertensives, antagonists of thealpha-2-adrenergic receptor, inhibitors of neutral endopeptidase,thrombocyte aggregation inhibitors and others or combinations thereofare suitable.

In another aspect, this invention relates to the use of a compoundaccording to the invention or a physiologically acceptable salt thereofcombined with at least one of the active substances described above as acombination partner, for preparing a medicament which is suitable forthe treatment or prevention of diseases or conditions which can beaffected by binding to the receptors for GLP-1 and glucagon and bymodulating their activity. This is preferably a disease in the contextof the metabolic syndrome, particularly one of the diseases orconditions listed above, most particularly diabetes or obesity orcomplications thereof.

The use of the compounds according to the invention, or aphysiologically acceptable salt thereof, in combination with one or moreactive substances may take place simultaneously, separately orsequentially.

The use of the compound according to the invention, or a physiologicallyacceptable salt thereof, in combination with another active substancemay take place simultaneously or at staggered times, but particularlywithin a short space of time. If they are administered simultaneously,the two active substances are given to the patient together; if they areused at staggered times, the two active substances are given to thepatient within a period of less than or equal to 12 hours, butparticularly less than or equal to 6 hours.

Consequently, in another aspect, this invention relates to a medicamentwhich comprises a compound according to the invention or aphysiologically acceptable salt of such a compound and at least one ofthe active substances described above as combination partners,optionally together with one or more inert carriers and/or diluents.

The compound according to the invention, or physiologically acceptablesalt or solvate thereof, and the additional active substance to becombined therewith may both be present together in one formulation, forexample a tablet or capsule, or separately in two identical or differentformulations, for example as so-called kit-of-parts.

LEGENDS TO THE FIGURES

FIG. 1. Comparison of Dynamic Debye plots for peptide formulationsexhibiting reversible self-association (attractive interactions) andrepulsive virial interactions

FIG. 2. SEQ ID NO: 6, SEQ ID NO: 7—Blood glucose profile following firsttreatment in fed DIO mice

FIG. 3. SEQ ID NO: 6, SEQ ID NO: 7—Body mass in DIO mice

FIG. 4. SEQ ID NO: 6, SEQ ID NO: 7—Body mass change in DIO mice

FIG. 5. SEQ ID SEQ ID NO: 6, SEQ ID NO: 7—Whole body fat mass change inDIO mice (n=8/group, n=7 DIO-Vehicle)

FIG. 6. SEQ ID NO: 6, SEQ ID NO: 7—Terminal liver mass in DIO mice

FIG. 7. SEQ ID NO: 6, SEQ ID NO: 7—Terminal plasma alanineaminotransferase (ALAT) concentrations in DIO mice (n=8/group, n=7DIO-Vehicle)

FIG. 8. SEQ ID NO: 6, SEQ ID NO: 7—Terminal total plasma cholesterolconcentrations in DIO mice

FIG. 9. SEQ ID NO: 6, SEQ ID NO: 7—Terminal plasma insulinconcentrations in DIO mice

FIG. 10. SEQ ID NO: 6, SEQ ID NO: 7—Blood glucose profile followingsingle treatment in fed db/db mice

FIG. 11. SEQ ID NO: 6, SEQ ID NO: 7—Blood glucose area under the curve(AUC) in fed db/db mice

FIG. 12. SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10—Blood glucose profilefollowing first treatment in fed DIO mice

FIG. 13. SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10—Body mass in DIOmice

FIG. 14. SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10—Body mass changein DIO mice

FIG. 15. SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10—Whole body fatmass change in DIO mice

FIG. 16. SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10—Terminal livermass in DIO mice

FIG. 17. SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10—Terminal plasmaalanine aminotransferase concentrations in DIO mice

FIG. 18. SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10—Terminal totalplasma cholesterol concentrations in DIO mice

FIG. 19. SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10—Terminal plasmainsulin concentrations in DIO mice

FIG. 20. SEQ ID NO: 13—Blood glucose profile following single treatmentin fed db/db mice

FIG. 21. SEQ ID NO: 13—Blood glucose area under the curve (AUC) in feddb/db mice

METHODS Abbreviations Employed are as Follows:

-   AA amino acid-   AEEAc (2-(2-aminoethoxy)ethoxy)acetyl-   Aib alpha-amino-isobutyric acid-   cAMP cyclic adenosine monophosphate-   Boc tert-butyloxycarbonyl-   BOP (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium    hexafluorophosphate-   BSA bovine serum albumin-   tBu tertiary butyl-   dAla D-alanine-   DCM dichloromethane-   Dde 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-ethyl-   ivDde 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methyl-butyl-   DIC N,N′-diisopropylcarbodiimide-   DIPEA N,N-diisopropylethylamine-   DMEM Dulbecco's modified Eagle's medium-   DMF dimethyl formamide-   DMS dimethylsulfide-   EDT ethanedithiol-   FA formic acid-   FBS fetal bovine serum-   Fmoc fluorenylmethyloxycarbonyl-   gAAA gamma-amino adipic acid-   gGlu gamma-glutamate (γE)-   HATU O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium    hexafluorophosphate-   HBSS Hanks' Balanced Salt Solution-   HBTU 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium    hexafluorophosphate-   HEPES 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid-   HOBt 1-hydroxybenzotriazole-   HOSu N-hydroxysuccinimide-   HPLC High Performance Liquid Chromatography-   HTRF Homogenous Time Resolved Fluorescence-   IBMX 3-isobutyl-1-methylxanthine-   LC/MS Liquid Chromatography/Mass Spectrometry-   Mrnt monomethoxy-trityl-   Palm palmitoyl-   PBS phosphate buffered saline-   PEG polyethylene glycole-   PK pharmacokinetic-   RP-HPLC reversed-phase high performance liquid chromatography-   Stea stearyl-   TFA trifluoroacetic acid-   Trt trityl-   UV ultraviolet

General Synthesis of Peptidic Compounds Materials

Different Rink-Amide resins(4-(2′,4′-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucylaminomethylresin, Merck Biosciences;4-[(2,4-Dimethoxyphenyl)(Fmoc-amino)methyl]phenoxy acetamido methylresin, Agilent Technologies) were used for the synthesis of peptideamides with loadings in the range of 0.2-0.7 mmol/g.

Fmoc protected natural amino acids were purchased from ProteinTechnologies Inc., Senn Chemicals, Merck Biosciences, Novabiochem, IrisBiotech, Bachem, Chem-Impex International or MATRIX Innovation. Thefollowing standard amino acids were used throughout the syntheses:Fmoc-L-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-L-Asn(Trt)-OH,Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Cys(Trt)-OH, Fmoc-L-Gln(Trt)-OH,Fmoc-L-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Ile-OH,Fmoc-L-Leu-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Met-OH, Fmoc-L-Phe-OH,Fmoc-L-Pro-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Thr(tBu)-OH,Fmoc-L-Trp(Boc)-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Val-OH.

In addition, the following special amino acids were purchased from thesame suppliers as above: Fmoc-L-Lys(ivDde)-OH, Fmoc-L-Lys(Mmt)-OH,Fmoc-Aib-OH, Fmoc-D-Ser(tBu)-OH, Fmoc-D-Ala-OH, Boc-L-His(Boc)-OH(available as toluene solvate) and Boc-L-His(Trt)-OH.

Furthermore, the building blocks(2S)-6-[[(4S)-5-tert-butoxy-4-[[(4S)-5-tert-butoxy-4-(hexadecanoylamino)-5-oxo-pentanoyl]amino]-5-oxo-pentanoyl]amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoicacid and Boc-L-His(Trt)-Aib-OH can be applied. Both building blocks aresynthesized separately.

The solid phase peptide syntheses were performed for example on aPrelude Peptide Synthesizer (Protein Technologies Inc) or similarautomated synthesizer using standard Fmoc chemistry and HBTU/DIPEAactivation. DMF was used as the solvent. Deprotection: 20%piperidine/DMF for 2×2.5 min. Washes: 7×DMF. Coupling 2:5:10 200 mMAA/500 mM HBTU/2M DIPEA in DMF 2× for 20 min. Washes: 5×DMF.

In cases where a Lys-side-chain was modified, Fmoc-L-Lys(ivDde)-OH orFmoc-L-Lys(Mmt)-OH was used in the corresponding position. Aftercompletion of the synthesis, the ivDde group was removed according to amodified literature procedure (S. R. Chhabra et al., Tetrahedron Lett.,1998, 39, 1603), using 4% hydrazine hydrate in DMF. The Mmt group wasremoved by repeated treatment with AcOH/TFE/DCM (1/2/7) for 15 minutesat RT, the resin then repeatedly washed with DCM, 5% DIPEA in DCM and 5%DIPEA in DCM/DMF.

The following acylations were carried out by treating the resin with theN-hydroxy succinimide esters of the desired acid or using couplingreagents like HBTU/DIPEA or HOBt/DIC.

All the peptides that have been synthesized were cleaved from the resinwith King's cleavage cocktail consisting of 82.5% TFA, 5% phenol, 5%water, 5% thioanisole, 2.5% EDT. The crude peptides were thenprecipitated in diethyl or diisopropyl ether, centrifuged, andlyophilized. Peptides were analyzed by analytical HPLC and checked byESI mass spectrometry. Crude peptides were purified by a conventionalpreparative RP-HPLC purification procedure.

Alternatively, peptides were synthesized by a manual synthesisprocedure: 0.3 g Desiccated Rink amide MBHA Resin (0.66 mmol/g) wasplaced in a polyethylene vessel equipped with a polypropylene filter.Resin was swollen in DCM (15 ml) for 1 h and DMF (15 ml) for 1 h. TheFmoc group on the resin was de-protected by treating it twice with 20%(v/v) piperidine/DMF solution for 5 and 15 min. The resin was washedwith DMF/DCM/DMF (6:6:6 time each). A Kaiser test (quantitative method)was used for the conformation of removal of Fmoc from solid support. TheC-terminal Fmoc-amino acid (5 equiv. excess corresponding to resinloading) in dry DMF was added to the de-protected resin and coupling ofthe next Fmoc-amino acid was initiated with 5 equivalent excess of DICand HOBT in DMF. The concentration of each reactant in the reactionmixture was approximately 0.4 M. The mixture was rotated on a rotor atroom temperature for 2 h. Resin was filtered and washed with DMF/DCM/DMF(6:6:6 time each). Kaiser test on peptide resin aliquot upon completionof coupling was negative (no colour on the resin). After the first aminoacid attachment, the unreacted amino group, if any, in the resin wascapped used acetic anhydride/pyridine/DCM (1:8:8) for 20 minutes toavoid any deletion of the sequence. After capping, resin was washed withDCM/DMF/DCM/DMF (6/6/6/6 time each). The Fmoc group on the C-terminalamino acid attached peptidyl resin was deprotected by treating it twicewith 20% (v/v) piperidine/DMF solution for 5 and 15 min. The resin waswashed with DMF/DCM/DMF (6:6:6 time each). The Kaiser test on peptideresin aliquot upon completion of Fmoc-deprotection was positive.

The remaining amino acids in target sequence on Rink amide MBHA Resinwere sequentially coupled using Fmoc AA/DIC/HOBt method using 5equivalent excess corresponding to resin loading in DMF. Theconcentration of each reactant in the reaction mixture was approximately0.4 M. The mixture was rotated on a rotor at room temperature for 2 h.Resin was filtered and washed with DMF/DCM/DMF (6:6:6 time each). Aftereach coupling step and Fmoc deprotection step, a Kaiser test was carriedout to confirm the completeness of the reaction.

After the completion of the linear sequence, the ε-amino group of lysineused as branching point or modification point was deprotected by using2.5% hydrazine hydrate in DMF for 15 min×2 and washed with DMF/DCM/DMF(6:6:6 time each). The γ-carboxyl end of glutamic acid was attached tothe ε-amino group of Lys using Fmoc-Glu(OH)-OtBu with DIC/HOBt method (5equivalent excess with respect to resin loading) in DMF. The mixture wasrotated on a rotor at room temperature for 2 h. The resin was filteredand washed with DMF/DCM/DMF (6×30 ml each). The Fmoc group on theglutamic acid was de-protected by treating it twice with 20% (v/v)piperidine/DMF solution for 5 and 15 min (25 ml each). The resin waswashed with DMF/DCM/DMF (6:6:6 time each). A Kaiser test on peptideresin aliquot upon completion of Fmoc-deprotection was positive.

If the side-chain branching also contains one more γ-glutamic acid, asecond Fmoc-Glu(OH)-OtBu used for the attachment to the free amino groupof γ-glutamic acid with DIC/HOBt method (5 equivalent excess withrespect to resin loading) in DMF. The mixture was rotated on a rotor atroom temperature for 2 h. Resin was filtered and washed with DMF/DCM/DMF(6×30 ml each). The Fmoc group on the γ-glutamic acid was de-protectedby treating it twice with 20% (v/v) piperidine/DMF solution for 5 and 15min (25 mL). The resin was washed with DMF/DCM/DMF (6:6:6 time each). AKaiser test on peptide resin aliquot upon completion ofFmoc-deprotection was positive.

Palmitic Acid and Stearic Acid Attachment to Side Chains of GlutamicAcid:

To the free amino group of γ-glutamic acid, palmitic acid or stearicacid (5 equiv.) dissolved in DMF was added and coupling was initiated bythe addition of DIC (5 equiv.) and HOBt (5 equiv.) in DMF. The resin waswashed with DMF/DCM/DMF (6:6:6 time each).

Final Cleavage of Peptide from the Resin:

The peptidyl resin synthesized by manual synthesis was washed with DCM(6×10 ml), MeOH (6×10 ml) and ether (6×10 ml) and dried in vacuumdesiccators overnight. The cleavage of the peptide from the solidsupport was achieved by treating the peptide-resin with reagent cocktail(80.0% TFA/5% thioanisole/5% phenol/2.5% EDT, 2.5% DMS and 5% DCM) atroom temperature for 3 h. Cleavage mixture was collected by filtrationand the resin was washed with TFA (2 ml) and DCM (2×5 ml). The excessTFA and DCM was concentrated to small volume under nitrogen and a smallamount of DCM (5-10 ml) was added to the residue and evaporated undernitrogen. The process was repeated 3-4 times to remove most of thevolatile impurities. The residue was cooled to 0° C. and anhydrous etherwas added to precipitate the peptide. The precipitated peptide wascentrifuged and the supernatant ether was removed and fresh ether wasadded to the peptide and re-centrifuged. The crude sample waspreparative HPLC purified and lyophilized. The identity of peptide wasconfirmed by LCMS.

In addition, a different route for the introduction of the lysine sidechain is used, applying a prefunctionalized building block where theside chain is already attached to the lysine (e.g.(2S)-6-[[(4S)-5-tert-butoxy-4-[[(4S)-5-tert-butoxy-4-(hexadecanoylamino)-5-oxo-pentanoyl]amino]-5-oxo-pentanoyl]amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoicacid) as coupling partner in the peptide synthesis. 0.67 mmol of peptideresin bearing an amino-group is washed with 20 ml of dimethylformamide.2.93 g of(2S)-6-[[(4S)-5-tert-butoxy-4-[[(4S)-5-tert-butoxy-4-(hexadecanoylamino)-5-oxo-pentanoyl]amino]-5-oxo-pentanoyl]amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoicacid is dissolved in 20 ml of dimethylformamide together with 310 mg ofhydroxybenzotriazol hydrate and 0.32 ml of diisopropylcarbodiimide.After stirring of 5 minutes the solution is added to the resin. Theresin is agitated for 20 h and then washed 3 times with 20 ml ofdimethylformamide each. A small resin sample is taken and subjected tothe Kaiser-test and the Chloranil-test (E. Kaiser, R. L. Colescott, C.D. Bossinger, P. I. Cook, Anal. Biochem. 1970, 34, 595-598;Chloranil-Test: T. Vojkovsky, Peptide Research 1995, 8, 236-237). Thisprocedure avoids the need of a selective deprotection step as well asthe selective attachment of the side chain building blocks on a veryadvanced synthesis intermediate.

Analytical HPLC/UPLC Method A: Detection at 210-225 nm

-   column: Waters ACQUITY UPLC® CSH™ C18 1.7 μm (150×2.1 mm) at 50° C.-   solvent: H₂O+0.05% TFA: ACN+0.035% TFA (flow 0.5 ml/min)-   gradient: 80:20 (0 min) to 80:20 (3 min) to 25:75 (23 min) to 2:98    (23.5 min) to 2:98 (30.5 min) to 80:20 (31 min) to 80:20 (37 min)-   optionally with mass analyzer: LCT Premier, electrospray positive    ion mode

Method B: Detection at 214

-   column: Waters ACQUITY UPLC® CSH™ C18 1.7 μm (150×2.1 mm) at 50° C.-   solvent: H₂O+0.05% TFA: ACN+0.035% TFA (flow 0.5 ml/min)-   gradient: 80:20 (0 min) to 80:20 (3 min) to 25:75 (23 min) to 2:98    (23.5 min) to 2:98 (30.5 min) to 80:20 (31 min) to 80:20 (37 min)-   optionally with mass analyzer: Agilent 6230 Accurate-Mass TOF, Dual    Agilent Jet Stream ESI

Method C: Detection at 214 nm

-   column: Waters ACQUITY UPLC® CSH™ C18 1.7 μm (150×2.1 mm) at 50° C.-   solvent: H₂O+0.1% TFA: ACN+0.1% TFA (flow 0.5 ml/min)-   gradient: 80:20 (0 min) to 80:20 (3 min) to 25:75 (23 min) to 2:98    (23.5 min) to 2:98 (30.5 min) to 80:20 (31 min) to 80:20 (38 min)-   optionally with mass analyzer: Agilent 6230 Accurate-Mass TOF,    Agilent Jet Stream ESI

Method D: Detection at 220 nm

-   column: Waters ACQUITY BEH C18 (2.1×100 mm×1.7 μm), Temp: 40° C.    Aries peptide XB C18 (4.6×250 mm×3.6 μm), Temp: 40° C.-   solvent: H₂O+0.1% formic acid (buffer A): ACN+0.1% formic acid (flow    1 ml/min) (buffer B)-   gradient: Equilibration of the column with 2% buffer B and elution    by a gradient of 2% to 70% buffer B during 15 min??

Method E: Detection at 215 nm

-   column: Waters ACQUITY UPLC® CSH™ C18 1.7 μm (150×2.1 mm) at 50° C.-   solvent: H₂O+0.05% TFA: ACN+0.035% TFA (flow 0.5 ml/min)-   gradient: 80:20 (0 min) to 80:20 (3 min) to 25:75 (23 min) to 5:95    (23.5 min) to 5:95 (25.5 min) to 80:20 (26 min) to 80:20 (30 min)

General Preparative HPLC Purification Procedure

The crude peptides were purified either on an Akta Purifier System, aJasco semiprep HPLC System or a Agilent 1100 HPLC system. PreparativeRP-C18-HPLC columns of different sizes and with different flow rateswere used depending on the amount of crude peptide to be purified.Acetonitrile+0.1% TFA (B) and water+0.1% TFA (A) were employed aseluents. Product-containing fractions were collected and lyophilized toobtain the purified product, typically as TFA salt.

Solubility Assessment of Exendin-4 Derivatives

Prior to the solubility measurement of a peptide batch, its purity wasdetermined through UPLC/MS.

For solubility testing the target concentration was 10 mg purecompound/ml. Therefore solutions from solid samples were prepared in abuffer systems with a concentration of 10 mg/mL compound based on thepreviously determined % purity:

Solubility buffer system A) Acetate Buffer pH 4.5, 100 mM sodium acetatetrihydrate, 2.7 mg/ml m-Cresol

Solubility buffer system B) Phosphate Buffer pH 7.4, 100 mM sodiumhydrogen phosphate, 2.7 mg/ml m-Cresol

Solubility buffer system C) Citrate Buffer pH6.0, citric acid 100 mM,2.7 mg/mL m-cresol

UPLC-UV was performed after 1 hour of gentle agitation from thesupernatant, which was obtained after 15 min of centrifugation at 2500RCF (relative centrifugal acceleration).

The solubility was determined by the comparison of the UV peak area of 2μL-injection of a buffered sample diluted 1:10 with a standard curve ofa reference peptide with known concentration. The different UVextinction coefficients of sample and reference peptide were calculatedbased on the different amino acid sequences and considered in theconcentration calculation.

Chemical Stability Assessment of Exendin-4 Derivatives

Prior to the chemical stability measurement of a peptide batch, itspurity was determined through UPLC/MS. For stability testing the targetconcentration was 1 mg pure compound/ml. Therefore solutions from solidsamples were prepared in a buffer system with a concentration of 1 mg/mLcompound based on the previously determined % purity:

Chemical stability buffer system A) 25 mM acetate buffer pH4.5, 3 mg/mLL-Methionine, 2.7 mg/mL m-cresol, 18 mg/mL Glycerol 85%

Chemical stability buffer system B) 25 mM phosphate buffer pH6.0, 3mg/mL L-Methionine, 2.7 mg/mL m-cresol, 18 mg/mL Glycerol 85%

Peptide solutions were filtered through 0.22 μM pore size and filledinto aliquots under aseptic conditions. At starting point UPLC-UV wasperformed by injection of 2 μl of the undiluted sample.

For chemical stability testing, aliquots were stored for 28 days at Sand40° C. After this time course the samples were centrifuged for 15 min at2500 RCF. Then 2 μl of the undiluted supernatant were analysed withUPLC-UV.

The chemical stability was rated through the relative loss of puritycalculated by the equation:

[(purity at starting point)−(purity after 28 days at X° C.)]/(purity atstarting point)]*100%

X=5 or 40° C.

The purity is calculated as

[(peak area peptide)/(total peak area)]*100%

Dynamic Light Scattering (DLS) for the Assessment of Physical Stability

A monochromatic and coherent light beam (laser) is used to illuminatethe liquid sample. Dynamic Light Scattering (DLS) measures lightscattered from particles (1 nm≤radius≤1 μm) that undergo Brownianmotion. This motion is induced by collisions between the particles andsolvent molecules that themselves are moving due to their thermalenergy. The diffusional motion of the particles results in temporalfluctuations of the scattered light [Pecora, R. Dynamic LightScattering: Applications of Photon Correlation Spectroscopy, PlenumPress, 1985].

The scattered light intensity fluctuations are recorded and transformedinto an autocorrelation function. By fitting the autocorrelation curveto an exponential function, the diffusion coefficient D of the particlesin solution can be derived. The diffusion coefficient is then used tocalculate the hydrodynamic radius R_(h) (or apparent Stokes radius)through the Stokes-Einstein equation assuming spherical particles. Thiscalculation is defined in ISO 13321 and ISO 22412 [InternationalStandard ISO13321 Methods for Determination of Particle SizeDistribution Part 8: Photon Correlation Spectroscopy, InternationalOrganisation for Standardisation (ISO) 1996; International StandardISO22412 Particle Size Analysis—Dynamic Light Scattering, InternationalOrganisation for Standardisation (ISO) 2008].

In case of polydisperse samples, the autocorrelation function is the sumof the exponential decays corresponding to each of the species. Thetemporal fluctuations of the scattered light can then be used todetermine the size distribution profile of the particle fraction orfamily. The first order result is an intensity distribution of scatteredlight as a function of the particle size. The intensity distribution isnaturally weighted according to the scattering intensity of eachparticle fraction or family. For biological materials or polymers theparticle scattering intensity is proportional to the square of themolecular weight. Thus, small amount of aggregates/agglomerates orpresence or a larger particle species can dominate the intensitydistribution. However this distribution can be used as a sensitivedetector for the presence of large material in the sample. The intensitydistribution can be converted into a volume or mass distribution of theparticle sizes using the Mie theory under certain assumptions. Incontrast to the intensity distribution, the mass distribution is bestused for comparative purposes and should never be considered absolute(due to the underlying assumptions).

The DLS technique produces distributions with inherent peak broadening.The polydispersity index % Pd is a measure of the width of the particlesize distribution and is calculated by standard methods described inISO13321 and ISO22412 [International Standard ISO13321 Methods forDetermination of Particle Size Distribution Part 8: Photon CorrelationSpectroscopy, International Organisation for Standardisation (ISO) 1996;International Standard ISO22412 Particle Size Analysis—Dynamic LightScattering, International Organisation for Standardisation (ISO) 2008].

DLS Interaction Parameter (k_(D))

The DLS interaction parameter (k_(D)) is a measure to describeinter-particle interactions, where the particles are folded proteins orpeptides [Sandeep Yadav et al. (2009) J Pharm Sc, Vol 99(3), pp1152-1168; Brian D. Connolly et al. (2012) Biophysical Journal Volume103, pp 69-78].

The parameter k_(D) is derived from the concentration dependence of thediffusion coefficient D, which is given by an expansion in powers of theconcentration c:

D(c)=D ₀(1+k _(D) c+k _(iD) c ² +k _(jD) c ³+ . . . )

Neglecting the higher order terms, i.e. k_(iD)=k_(jD)= . . . =0, thedata can be fitted linearly and k_(D) is obtained from the slope of thecurve D=D₀ (1+k_(D)c) and D₀. D₀ is the diffusion coefficient at zeroconcentration. The parameter k_(D) can be used to describe interactionof protein or peptide molecules or oligomers with their-self and theirenvironment in solution and is theoretically related to the virialcoefficient B₂₂ as for example described by Harding and Johnson, where Mis the molar mass, k_(s) the first order concentration coefficient ofsedimentation velocity and u the partial specific volume [Harding S E,Johnson P. (1985) Biochem J, 231, pp 543-547].

k _(D)=2B ₂₂ M−k _(s) −u

Positive B₂₂ values indicate samples that favor salvation overself-association, while negative B₂₂ values indicate samples that preferself-association. From a pragmatic point of view, k_(D) is analogous inits meaning to B₂₂ and gives information on the net-forces between themolecules. High values indicate strong net-repulsive interactions, whilelow values indicate net-attractive forces. Therefore, k_(D) can be usedfor relative, qualitative comparison (see FIG. 1).

For every peptide solution, the hydrodynamic radius R_(h) and thediffusion constant D (related via the Stokes-Einstein equation) weredetermined as an average over triplicates. Both parameters weredetermined at different concentrations (e.g., R_(h1) and D₁: 1 mg/ml andR_(h5) and D₅: 5 mg/ml) in the same buffer system. The difference ofthese parameters between low and high peptide concentration is asurrogate for the DLS interaction parameter k_(D). R_(h5)<R_(h1) orD₅>D₁ correspond to k_(D)>0 and therefore to repulsive inter-particleinteractions that result in improved physical (or colloidal) stability.

DLS buffer system A) 25 mM acetate buffer pH4.5, 3 mg/mL L-Methionine,2.7 mg/mL m-cresol, 18 mg/mL Glycerol 85%

DLS buffer system B) 25 mM phosphate buffer pH6.0, 3 mg/mL L-Methionine,2.7 mg/mL m-cresol, 18 mg/mL Glycerol 85%

DLS Method A: DLS measurements were performed on a W130i apparatus (AvidNano Ltd, High Wycombe, UK) and using a low-volume disposable cuvette(UVette, Eppendorf AG, Hamburg, Germany). The data were processed withi-Size 3.0 provided by Avid Nano. Parameters of the particle sizedistribution were determined with non-negatively constrained leastsquares (NNLS) methods using DynaLS algorithms. Measurements were takenat 25° C. with a 660 nm laser light source and at an angle of 90°.

DLS Method B: DLS measurements were performed on a Nanosizer ZS (MalvernInstruments, Malvern, UK) and using disposible UV cuvettes (Brand macro,2.5 mL and Brand semi-micro 1.5 mL, Brand GmbH+Co KG, Wertheim,Germany). The data were processed with Malvern Zetasizer softwareVersion 7.10 or 7.01. Parameters of the particle size distribution weredetermined with non-negatively constrained least squares (NNLS) methods.Measurements were taken at 25° C. with a 633 nm laser light source inNIBS (Non-Invasive Back-Scatter) mode at an angle of 173°.

DLS Method C: DLS measurements were performed on a DynaPro Plate ReaderII (Wyatt Technology, Santa Barbara, Calif., US) and using one of thefollowing black, low volume, and non-treated plates: polystyrene 384assay plate with clear bottom (Corning, N.Y., US), polystyrene 96 assayplate with clear bottom (Corning, N.Y., US), cyclo olefin polymer (COP)384 assay plate with clear bottom (Aurora, Mont., US), or polystyrene384 assay plate with clear bottom (Greiner Bio-One, Germany). The datawere processed with the Dynamics software provided by Wyatt Technology.Parameters of the particle size distribution were determined withnon-negatively constrained least squares (NNLS) methods using DynaLSalgorithms. Measurements were taken at 25° C. with an 830 nm laser lightsource at an angle of 158°.

ThT Assay for the Assessment of Physical Stability

Low physical stability of a peptide solution may lead to amyloid fibrilformation, which is observed as well-ordered, thread-like macromolecularstructures in the sample, which eventually may lead to gel formation.Thioflavin T (ThT) is widely used to visualize and quantify the presenceof misfolded protein aggregates. [Biancalana et al. (2010) Biochimica etBiophysica Acta. 1804 (7): 1405-1412.]. When it binds to fibrils, suchas those in amyloid aggregates, the dye displays a distinct fluorescencesignature [Naiki et al. (1989) Anal. Biochem. 177, 244-249; LeVine(1999) Methods. Enzymol. 309, 274-284]. The time course for fibrilformation often follows the characteristic shape of a sigmoidal curveand can be separated into three regions: a lag phase, a fast growthphase, and a plateau phase.

The typical fibril formation process starts with the lag phase in whichthe amount of partially folded peptide turned into fibrils is notsignificant enough to be detected. The lag-time corresponds to the timethe critical mass of the nucleus is built. Afterwards, a drasticelongation phase follows and fibril concentration increases rapidly.

Investigations were carried out to determine fibrillation tendenciesunder stress conditions by shaking at 37° C. within Fluoroskan AscentFL.

For the tests in Fluoroskan Ascent FL, 200 μL sample were placed into a96 well mictrotiter plate PS, flat bottom, Greiner Fluotrac No. 655076.Plates were sealed with Scotch Tape (Quiagen). Samples were stressed bycontinuous cycles of 10 s shaking at 960 rpm and 50 s rest period at 37°C. The kinetic was monitored by measuring fluorescence intensity every20 minutes.

Peptides were diluted in a buffer system to a final concentration of 3mg/ml. 20 μL of a 10.1 mM ThT solution in H₂O were added to 2 mL ofpeptide solution to receive a final concentration of 100 μM ThT. Foreach sample eight replicates were tested.

Tht buffer system A) 100 mM Acetate pH 4.5 including m-cresol (100 mMNatriumacetat trihydrat, pH adjustment using 2N CH3COOH, 2.7 mg/mLm-cresol) Tht buffer system B) 100 mM citrate buffer pH 6.0

In Vitro Cellular Assays for GLP-1, Glucagon and GIP Receptor Efficacy

Agonism of compounds for the receptors was determined by functionalassays measuring cAMP response of HEK-293 cell lines stably expressinghuman GLP-1, GIP or glucagon receptor.

cAMP content of cells was determined using a kit from Cisbio Corp. (cat.no. 62AM4PEJ) based on HTRF (Homogenous Time Resolved Fluorescence). Forpreparation, cells were split into T175 culture flasks and grownovernight to near confluency in medium (DMEM/10% FBS). Medium was thenremoved and cells washed with PBS lacking calcium and magnesium,followed by proteinase treatment with accutase (Sigma-Aldrich cat. no.A6964). Detached cells were washed and resuspended in assay buffer(1×HBSS; 20 mM HEPES, 0.1% BSA, 2 mM IBMX) and cellular densitydetermined. They were then diluted to 400000 cells/ml and 25 μl-aliquotsdispensed into the wells of 96-well plates. For measurement, 25 μl oftest compound in assay buffer was added to the wells, followed byincubation for 30 minutes at room temperature. After addition of HTRFreagents diluted in lysis buffer (kit components), the plates wereincubated for 1 hr, followed by measurement of the fluorescence ratio at665/616 nm. In vitro potency of agonists was quantified by determiningthe concentrations that caused 50% activation of maximal response(EC50).

Bioanalytical Screening Method for Quantification of Exendin-4Derivatives in Mice and Pigs

Mice were dosed 1 mg/kg subcutaneously (s.c.). The mice were sacrificedand blood samples were collected after 0.25, 0.5, 1, 2, 4, 8, 16 and 24hours post application. Plasma samples were analyzed after proteinprecipitation via liquid chromatography mass spectrometry (LC/MS). PKparameters and half-life were calculated using WinonLin Version 5.2.1(non-compartment model).

Female Göttinger minipigs were dosed 0.05 mg/kg, 0.075 mg/kg or 0.1mg/kg subcutaneously (s.c.). Blood samples were collected after 0.25,0.5, 1, 2, 4, 8, 24, 32, 48, 56 and 72 hours post application. Plasmasamples were analyzed after protein precipitation via liquidchromatography mass spectrometry (LC/MS). PK parameters and half-lifewere calculated using WinonLin Version 5.2.1 (non-compartment model).

Acute and Chronic Effects after Subcutaneous Treatment on Blood Glucose,Body Mass, Whole Body Fat Content, and Feed Consumption in FemaleDiet-Induced Obese (DIO) C57BL/6 Mice

Female C57BL/6NHsd mice were ordered group housed from Envigo RMS Inc.,shipped group housed and remained group housed with shipped cage matesin shoebox caging with wood chip bedding until day 38 of the predosephase. At the study start mice were between 25-26 weeks old.

Mice were housed under vivarium conditions that included a 12 hlight/dark cycle (light phase 04:00 AM-4:00 PM), room temperaturesbetween 23-26° C. and a relative humidity between 30-70%. All animalshad free access to water and a high fat diet (TD97366) for 16 weeksprior to pharmacological intervention (dosing phase). Feed was replacedwith fresh feed weekly until and for the last time on day 38 of thepredose phase. During the subsequent dosing phase, approximately 50% ofthe remaining feed were removed, replaced with fresh feed, and pelletswere mixed evenly once per week.

On predose day 38, obese DIO mice were assigned to treatment groups(n=8) to match mean body masses between all DIO groups. An age-matchedgroup with ad libitum access to a rodent maintenance diet (Teklad GlobalDiets Rodent 2014, pelleted) was included in the study as a lean controlgroup. In the predosing phase from day 32 through 38, all study animalswere treated with vehicle (Phosphate Buffered Saline, PBS, Gibco,without CaCl₂ and MgCl₂) once daily (s.c. approximately 0.2 mL/mouse).

On day 37 of the predose phase, the test article was diluted with PBS toa concentration of 100 μg/mL and aliquots of this stock solution werestored at approximately ≤−60° C. Stock aliquots were thawed for weeklyuse and thereafter stored in a refrigerator at approximately 4° C. Theinjected test article solution was prepared fresh once on each dosingday by diluting stock solution with PBS to achieve the desiredconcentration.

Mice were treated twice daily with a s.c. injection of PBS-vehicle orthe test article for 28 days. The morning dosing was initiated andcompleted between 06:00 and 07:30 AM and the afternoon dosing between2:00 and 3:30 PM. On day 28 of the dosing phase only the morning dosewas administered. The applied volume was 5 ml/kg and the dose wasadjusted to the most recent body mass recording of each individual.

1) Acute Effect on Blood Glucose Profiles in Fed, Female DIO Mice:

Animals had unlimited access to water and feed during the experiment. Onday 1 of the dosing phase approximately 5 μL of blood were collected viatail clips at hour 0, prior to the first s.c. dose of PBS-vehicle ortest-article as well as 1, 2, 3, 4, 6, and 24 hours post-dose. Animalsreceived the second s.c. dose of PBS-vehicle or test-article 8 hoursafter the first dose. The 24 hour blood collection of the blood glucoseprofile was performed prior to dosing on day 2. Glucose measurementswere performed in whole blood and in duplicate or triplicate using Avivaglucometers.

2) Chronic Effect on Body Mass in Fed, Female DIO Mice:

Body mass was measured daily approximately between 06:00-07:30 AM fromday 32 through 38 of the predosing phase and throughout the 28 days ofthe dosing phase. During the dosing phase mice were treated twice dailywith either a s.c. injection of PBS-vehicle or test article.

3) Chronic Effect on Whole Body Fat Mass in Fed, Female DIO Mice:

To determine whole body fat mass Quantitative Nuclear Magnetic Resonance(QNMR) measurements were performed on predosing day 37 and on day 26 ofthe dosing phase. During the dosing phase mice were treated twice dailywith either a s.c. injection of PBS-vehicle or test article.

4) Effect on Feed Consumption in Female DIO Mice:

Feed consumption was based on the daily assessment of the feeder weightsof each cage between 06:00-07:30 AM. Each cage housed four mice and feedconsumption was calculated throughout the 28 days of the dosing phase.During the dosing phase mice were treated twice daily with either a s.c.injection of PBS-vehicle or test article.

5) Terminal Plasma Parameters in Non-Fasting, Female DIO Mice:

On day 28 blood was collected prior to any other inlife activities fordetermination of plasma insulin concentrations. Then the morning dosewas administered and necropsy was performed 4 hours post-dose. For thispurpose animals were anesthetized with isoflurane and blood wascollected by orbital bleeding.

5) Terminal Liver Mass in Non-Fasting, Female DIO Mice:

On day 28 and 4 hours after the morning dose, blood was collected frommice under isoflurane anesthesia as described above. Then mice werekilled and livers were collected and weighed.

6) Quantification of Liver Lipids

Liver aliquots were incubated with dichlormethane:methanol (2:1). Thelipohilic and lipohobic phase were separated by adding dH₂O andsubsequent centrifugation. The bottom lipophilic phase was collected andthe procedure repeated with the remaining lipophobic layer and livertissue. Next the lipophilic phases were combined and the solventevaporated. At 60° C. and continuous shaking samples were then incubatedwith 2-propanol. Total cholesterol, triacylglycerol and phospholipidconcentrations were quantified enzymatically with a commercial kitaccording to the manufacturer's instructions.

7) Statistical Analyses:

Data are depicted as means±SEM. Statistical analyses were performed withSigmaplot 12.5. A One-Way ANOVA and Tukey Test were used for pairwisemultiple comparisons of all groups (n=8/group, deviations from groupsize are indicated). When the difference in the mean values of the twogroups was greater than 0.05 they were considered statisticallysignificantly different. Lean-Vehicle group data serve as a referencedataset for the non-obese state.

Acute Effects after Subcutaneous Treatment on Blood GlucoseConcentrations in Non-Fasting, Female, Diabetic Db/Db Mice

Female healthy, lean BKS.Cg-(lean)/OlaHsd and diabetes-prone, obeseBKS.Cg-+Lepr^(db)/+Lepr^(db)/OlaHsd mice were ordered group housed fromEnvigo RMS Inc., shipped group housed and remained group housed inshoebox caging with wood chip bedding until day 15 of the predose phase.At the study start mice were approximately 12 weeks old.

Mice were housed under vivarium conditions including a 12 h light/darkcycle (light phase 04:00 AM-4:00 PM), room temperatures between 23-26°C. and a relative humidity between 30-70%. All animals had free accessto water and Purina Fomulab Diet 5008.

On predose day 9 blood glucose and body mass (approximately between08:00-10:00 AM) as well as HbA1c measurements were performed. On day 15of the predose phase animals were assigned to treatment groups (n=8) andto new cages to match mean HbA1c and body masses between all db/dbgroups. An age-matched lean group was included in the study as ahealthy, lean reference.

Prior to day 1 of the dosing phase, the test article was diluted withPhosphate Buffered Saline (PBS, Gibco, without CaCl₂ and MgCl₂) to aconcentration of 1 mg/mL and aliquots of this stock solution were storedat approximately −60° C. On day 1 of the dosing phase the stock aliquotwas thawed and the injected test article solution was prepared fresh bydiluting it with PBS to achieve the desired concentration.

On Day 1 of the dosing phase db/db mice were either treated once with as.c. injection of PBS-vehicle or 30 μg/kg test article. The leanreference group was treated once with a s.c. injection of PBS-vehicle.The dosing was initiated and completed between 08:00 and 10:00 AM. Theapplied volume was 5 ml/kg and the dose was adjusted to the most recentbody mass recording of each individual.

1) Acute Effect on Blood Glucose Profiles in Non-Fasting Animals:

Animals had unlimited access to water and feed during the experiment. Onday 1 of the dosing phase approximately 5 μL of blood were collected viatail clips at minute −30 and 0 prior to any other inlife activities. Atminute 0 mice received a s.c. dose of either PBS-vehicle or 30 μg/kgtest article. Further blood samples were harvested at hour 0.25, 0.5, 1,1.5, 2, 3, 4, 6, 8, and 24 post-dose. Glucose measurements wereperformed in whole blood using AlphaTRAK glucometers. If the glucoseconcentrations of two measurements differed by more than 20 mg/dL athird value was recorded. The Area Under the Curve (AUC) for bloodglucose was calculated via the trapezoid method and for the duration ofthe 24 post-dose hours.

2) Statistical Analyses:

Data are depicted as means±SEM. Statistical analyses were performed withSigmaplot 12.5. A One-Way ANOVA and Tukey Test were used for pairwisemultiple comparisons of all groups (n=8/group, deviations from groupsize are indicated). When the difference in the mean values of the twogroups was greater than 0.05 they were considered statisticallysignificantly different. Data from a Non-Diabetic-Vehicle group serve asa reference dataset for the normoglycemic, lean state.

EXAMPLES

The invention is further illustrated by the following examples.

Example 1 Synthesis of SEQ ID NO: 6

The solid phase synthesis as described in Methods was carried out onNovabiochem Rink-Amide resin(4-(2′,4′-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucylaminomethylresin), 100-200 mesh, loading of 0.43 mmol/g. The Fmoc-synthesisstrategy was applied with HBTU/DIPEA-activation. In position 14Fmoc-Lys(Mmt)-OH and in position 1 Boc-His(Trt)-OH were used in thesolid phase synthesis protocol The Mmt-group was cleaved from thepeptide on resin as described in the Methods. HereafterPalm-gGlu-gGlu-OSu was coupled to the liberated amino-group employingDIPEA as base. The peptide was cleaved from the resin with King'scocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J. PeptideProtein Res. 1990, 36, 255-266). The crude product was purified viapreparative HPLC on a Waters column (Sunfire Prep C18 ODB 5 μm 50×150mm) using an acetonitrile/water gradient (both buffers with 0.1% TFA).The purified peptide was analysed by LCMS (Method B). Deconvolution ofthe mass signals found under the peak with retention time 9.824 minrevealed the peptide mass 4839.67 which is in line with the expectedvalue of 4839.67.

Example 2 Synthesis of SEQ ID NO: 7

The solid phase synthesis as described in Methods was carried out onNovabiochem Rink-Amide resin(4-(2′,4′-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucylaminomethylresin), 100-200 mesh, loading of 0.43 mmol/g. The Fmoc-synthesisstrategy was applied with HBTU/DIPEA-activation. In position 14Fmoc-Lys(Mmt)-OH and in position 1 Boc-His(Trt)-OH were used in thesolid phase synthesis protocol. The Mmt-group was cleaved from thepeptide on resin as described in the Methods. HereafterPalm-gGlu-gGlu-OSu was coupled to the liberated amino-group employingDIPEA as base. The peptide was cleaved from the resin with King'scocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J. PeptideProtein Res. 1990, 36, 255-266). The crude product was purified viapreparative HPLC on a Waters column (Sunfire Prep C18 OBD 5 μm 50×150mm) using an acetonitrile/water gradient (both buffers with 0.1% TFA).The purified peptide was analysed by LCMS (Method B).

Deconvolution of the mass signals found under the peak with retentiontime 9.935 min revealed the peptide mass 4853.73 which is in line withthe expected value of 4853.67.

Example 3 Synthesis of SEQ ID NO: 11

The solid phase synthesis as described in Methods was carried out onNovabiochem Rink-Amide resin(4-(2′,4′-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucylaminomethylresin), 100-200 mesh, loading of 0.43 mmol/g. The Fmoc-synthesisstrategy was applied with HBTU/DIPEA-activation. In position 14Fmoc-Lys(Mmt)-OH and in position 1 Boc-His(Trt)-OH were used in thesolid phase synthesis protocol. The Mmt-group was cleaved from thepeptide on resin as described in the Methods. HereafterPalm-gGlu-gGlu-OSu was coupled to the liberated amino-group employingDIPEA as base. The peptide was cleaved from the resin with King'scocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J. PeptideProtein Res. 1990, 36, 255-266). The crude product was purified viapreparative HPLC on a Waters column (Sunfire Prep C18 OBD 5 μm 50×150mm) using an acetonitrile/water gradient (both buffers with 0.1% TFA).The purified peptide was analysed by LCMS (Method B).

Deconvolution of the mass signals found under the peak with retentiontime 9.828 min revealed the peptide mass 4894.63 which is in line withthe expected value of 4894.64.

In an analogous way, the other peptides listed in Table 3 weresynthesized and characterized.

TABLE 3 list of synthesized peptides and comparison of calculated vs.found molecular weight SEQ ID calc. found Monoisotopic Retention NO Massmass or average mass time (min) 6 4839.7 4839.7 monoisotopic 9.824 74853.7 4853.7 monoisotopic 9.935 8 4880.6 4880.6 monoisotopic 9.585 94840.6 4840.6 monoisotopic 10.471 10 4854.6 4854.7 monoisotopic 10.64 114894.6 4849.6 monoisotopic 9.828 12 4879.7 4879.7 monoisotopic 9.490 134893.7 4893.7 monoisotopic 9.690 14 4710.6 4710.7 monoisotopic 9.435 154738.7 4783.7 monoisotopic 10.528 16 5024.7 5024.7 monoisotopic 9.137 175038.8 5038.8 monoisotopic 9.286 18 5025.7 5025.7 monoisotopic 9.573 195039.7 5039.7 monoisotopic 9.715 20 5041.8 5040.8 average 8.996 215055.8 5055.0 average 9.102 22 5042.8 5041.6 average 9.254 23 5056.85055.6 average 9.340

Example 4: Stability

Peptide samples were prepared in Chemical stability buffer system A andchemical stability was assessed as described in Methods. The results aregiven in Table 4.

TABLE 4 Stability SEQ ID Chemical stability [relative purity loss NO 28days 40° C.] (%) at pH 4.5 6 6.9 7 6.8 8 7.6 9 8.7 10 7.4 11 7.2 12 5.413 4.5 14 5.5 15 5.6 17 6.4 19 6.0 23 9.6

Example 5: Solubility

Peptide samples were prepared in solubility buffer system A andsolubility was assessed as described in Methods. The results are givenin Table 5.

TABLE 5 Solubility SEQ ID Solubility [mg/ml] NO at pH 4.5 6 >9.6 7 >9.98 >10 10 >10.1 11 >9.9 12 >9.3 13 >9.3 14 >9.3 15 >8.9 17 >9.2 19 >9.223 >10.4

Example 6: Stability as Assessed by DLS Interaction Parameter

The hydrodynamic radius radius R_(h) of peptide samples was determinedat different peptide concentrations (1 mg/ml and 5 mg/ml) in DLS buffersystem A using DLS method C as described in Methods as surrogate for theDLS interaction parameter k_(D). The results are given in Table 6.

TABLE 6 hydrodynamic radius R_(h) at peptide concentrations of 1 mg/mland 5 mg/ml. A decrease of R_(h) at the higher peptide concentrationindicates higher physical stability due to repulsive inter-particleinteractions. SEQ. R_(h1) [nm] R_(h5) [nm] Delta R_(h) ID c= 1 mg/ml c =5 mg/ml [nm] = R_(h5) − R_(h1) 6 2.3 1.8 −0.5 7 2.8 2.4 −0.3 13 2.8 2.5−0.3 14 2.7 2.4 −0.2 15 2.7 2.6 −0.2 17 2.9 2.4 −0.5 19 3.1 3.0 −0.1 232.8 2.7 −0.1

Example 7: Stability was Assessed in the ThT Assay

Lag time in hours in Thioflavin T (ThT) assay of peptide samples wasdetermined in Tht buffer system A as described in Methods. The resultsare given in Table 7.

TABLE 7 Lag time in hours in Thioflavin T (ThT) assay SEQ ID Increase inFl Lag time before NO at pH 4.5 increase [h] 6 NO >45 7 NO >45 8 NO >459 NO >45 10 NO >45 11 NO >45 12 NO >45 13 NO >45 16 NO >45 17 NO >45 18NO >45 19 NO >45 20 NO >45 21 NO >45 22 NO >45 23 NO >45

Example 8: In Vitro Data on Human GLP-1, Glucagon and GIP Receptor

Potencies of peptidic compounds at the GLP-1, glucagon and GIP receptorswere determined by exposing cells expressing human glucagon receptor(hGlucagon R), human GIP receptor (hGIP-R) or human GLP-1 receptor(hGLP-1 R) to the listed compounds at increasing concentrations andmeasuring the formed cAMP as described in Methods.

The results are shown in Table 8.

TABLE 8 EC50 values of exendin-4 derivatives at human GLP-1, Glucagonand GIP receptors (indicated in pM) SEQ ID NO EC50 hGLP-1R EC50hGlucagon R EC50 hGIP R 6 2.8 16.4 7.9 7 4.0 21.8 14.2 8 2.2 32.6 5.6 92.6 24.7 5.6 10 2.4 26.1 5.4 11 2.3 22.4 3.5 12 2.7 22.0 15.8 13 3.825.8 20.4 14 5.7 31.6 18.2 15 5.7 15.6 26.1 16 1.3 18.6 7.5 17 1.8 22.011.4 18 1.7 46.6 5.6 19 1.9 52.2 7.9 20 2.4 44.0 12.7 21 4.2 69.9 24.622 2.1 108.0 6.0 23 1.0 64.3 2.0

Example 9: Acute and Chronic Effects of SEQ ID NO: 6 and SEQ ID NO: 7after Subcutaneous Treatment on Blood Glucose and Body Weight in FemaleDiet-Induced Obese (DIO) C57BL/6NHsd Mice

Lean and diet-induced obese, 25-26 weeks old, female C57BL/6NHsd micewere treated twice daily with a s.c. injection of PBS, 30 μg/kg SEQ IDNO: 6, or 30 μg/kg SEQ ID NO: 7 for 28 days. The morning dosing wasinitiated and completed between 06:00 and 07:30 AM and the afternoondosing between 14:00 and 15:30 PM. The applied volume was 5 ml/kg andthe dose was adjusted to the most recent body mass recording of eachindividual.

1) Glucose Profile

Animals had unlimited access to water and feed during the experiment. Onday 1 of the dosing phase approximately 5 μL of blood were collected viatail clips at hour 0, prior to the first s.c. dose of PBS-vehicle, 30μg/kg SEQ ID NO: 6, or 30 μg/kg SEQ ID NO: 7, as well as 1, 2, 3, 4, 6,and 24 hours post-dose. Animals received the second s.c. dose ofPBS-vehicle, 30 μg/kg SEQ ID NO: 6, or 30 μg/kg SEQ ID NO: 7 8 hoursafter the first dose. The 24 hour blood collection of the blood glucoseprofile was performed prior to dosing on day 2. Glucose measurementswere performed in whole blood and in duplicate or triplicate using Avivaglucometers.

Both, 30 μg/kg SEQ ID NO: 6 and 30 μg/kg SEQ ID NO: 7 mediated acomparable decrease in blood glucose concentrations below concentrationsobserved in DIO control mice during the 24 hour blood glucose profile(FIG. 2).

2) Body Mass

Body mass was determined daily. In the dosing phase mice were treatedtwice daily with either a s.c. injection of PBS-vehicle, 30 μg/kg SEQ IDNO: 6, or 30 μg/kg SEQ ID NO: 7.

Chronic, twice daily treatment with SEQ ID NO: 6, or SEQ ID NO: 7resulted in a body mass reduction within the 28 days of the dosing phasein contrast to animals of the DIO-vehicle group (FIG. 3). Chronictreatment of DIO animals with 30 μg/kg SEQ ID NO: 6, or SEQ ID NO: 7induced a statistically significant decrease in body mass compared tothe DIO-Vehicle group (One-Way ANOVA and Tukey Test, FIG. 4, Table 9).

3) Whole Body Fat Mass

Whole body fat mass was determined via Nuclear Magnetic Resonance (QNMR)once in the predosing phase and once on day 26 of the dosing phasedaily. In the dosing phase mice were treated twice daily with either as.c. injection of PBS-vehicle, 30 μg/kg SEQ ID NO: 6, or 30 μg/kg SEQ IDNO: 7.

Chronic, twice daily treatment with SEQ ID NO: 6 and SEQ ID NO: 7resulted in a statistically significantly reduction in whole body fatmass within the 28 days compared to the DIO-Vehicle group (One-Way ANOVAand Tukey Test, FIG. 5, Table 9).

4) Terminal Liver Mass

On day 28 mice were euthanized 4 hours after the morning dose and liverswere collected.

Twice-daily, chronic treatment of DIO animals with 30 μg/kg SEQ ID NO:6, or 30 μg/kg SEQ ID NO: 7 resulted in a statistically significantlylower liver mass on day 28 of the dosing phase compared to DIO animalstreated with vehicle (One-Way ANOVA and Tukey Test, FIG. 6, Table 9).

5) Terminal Plasma Alanine Aminotransferase (ALAT) Concentration

On day 28 and 4 hours after the morning dose, blood was collected fromanesthetized, non-fasting mice by orbital bleeding.

Twice-daily, chronic treatment of DIO animals with 30 μg/kg SEQ ID NO: 6or 30 μg/kg SEQ ID NO: 7 resulted in a statistically significantly lowerplasma alanine aminotransferase concentration on day 28 of the dosingphase compared to DIO animals treated with vehicle suggestingimprovement of liver function (One-Way ANOVA and Tukey Test, FIG. 7,Table 9).

6) Terminal Plasma Total Cholesterol

On day 28 and 4 hours after the morning dose, blood was collected fromanesthetized, non-fasting mice by orbital bleeding.

Twice-daily, chronic treatment of DIO animals with 30 μg/kg SEQ ID NO: 6or 30 μg/kg SEQ ID NO: 7 resulted in a statistically significantreduction in plasma total cholesterol concentrations compared to DIOanimals treated with vehicle (One-Way ANOVA and Tukey Test, FIG. 8,Table 9).

7) Terminal Plasma Insulin

On day 28 and 4 hours after the morning dose, blood was collected fromanesthetized, non-fasting mice by orbital bleeding.

Twice-daily, chronic treatment of DIO animals with 30 μg/kg SEQ ID NO: 6or 30 μg/kg SEQ ID NO: 7 resulted in a statistically significantreduction in plasma insulin concentrations compared to DIO animalstreated with vehicle hinting towards an improvement of high fat-dietinduced insulin resistance (One-Way ANOVA and Tukey Test, FIG. 9, Table9).

8) Statistical Analyses

Data are depicted as means±SEM. Statistical analyses were performed withSigmaplot 12.5. A One-Way ANOVA and Tukey Test was used for pairwisemultiple comparisons of all groups (as indicated n=7-8/group). When thedifference in the mean values of the two groups was greater than 0.05they were considered statistically significantly different. Lean-Vehiclegroup data are depicted in the Figures serving as a reference datasetfor the non-obese state.

TABLE 9 Effects resulting from a 28 days qd dosing study with SEQ ID NO:6 and SEQ ID NO: 7 in fed, female diet-induced obese (DIO) C57BL/6 mice.Data are means ± SEM. One-Way ANOVA and Tukey Test, n = 8/group except n= 7 DIO-Vehicle. Example DIO-Vehicle DIO-SEQ ID NO: 6 DIO-SEQ ID NO: 7Dose twice-daily PBS twice-daily 30 μg/kg twice-daily 30 μg/kg Body mass 4.65 ± 0.33 −5.36 ± 0.36 −5.58 ± 0.60 change (g) P < 0.001 vs. P <0.001 vs. DIO-Vehicle DIO-Vehicle Whole body fat  3.37 ± 0.36 −4.33 ±0.42 −4.46 ± 0.50 mass change (g) P < 0.001 vs. P < 0.001 vs.DIO-Vehicle DIO-Vehicle Terminal liver  +1.94 ± 0.11 +1.16 ± 0.04 +1.14± 0.04 mass (g) P < 0.001 vs. P<0.001 vs. DIO-Vehicle DIO-VehicleTerminal plasma +155.25 ± 13.46 +62.25 ± 9.79  +47.63 ± 6.76  alanine P< 0.001 vs. P<0.001 vs. aminotransferase DIO-Vehicle DIO-Vehicle (U/L)Terminal plasma  +2.27 ± 0.09 +5.11 ± 0.12 +4.19 ± 0.15 totalcholesterol P < 0.001 vs. P < 0.001 vs. (mmol/L) DIO-Vehicle DIO-VehicleTerminal plasma +217.94 ± 51.48 +31.07 ± 5.26  +58.04 ± 6.33  insulin(U/L) P < 0.001 vs. P < 0.001 vs. DIO-Vehicle DIO-Vehicle

Example 10: Acute Effects of SEQ ID NO: 6 and SEQ ID NO: 7 afterSubcutaneous Treatment on Blood Glucose Concentrations in Non-Fasting,Female, Diabetic Db/Db Mice

Female, 12 weeks old, healthy, lean BKS.Cg-(lean)/OlaHsd ordiabetes-prone, obese BKS.Cg-+Leprdb/+Leprdb/OlaHsd mice were treatedonce with a s.c. injection of PBS, 30 μg/kg SEQ ID NO: 6 or 30 μg/kg SEQID NO: 7. The dosing was initiated and completed between 08:00 and 10:00AM. The applied volume was 5 ml/kg and the dose was adjusted to the mostrecent body mass recording of each individual. Diabetic db/db mice wereHbA1c- and body mass-matched. The lean reference group was treated oncewith a s.c. injection of PBS-vehicle.

1) Blood Glucose Profile

Animals had unlimited access to water and feed during the experiment. Onday 1 of the dosing phase approximately 5 μL of blood were collected viatail clips at minute −30 and 0 prior to any other inlife activities. Atminute 0 mice received a s.c. dose of either PBS-vehicle, 30 μg/kg SEQID NO: 6 or SEQ ID NO: 7. Further blood samples were harvested at minute15, 30, 60, 90, 120, 150, 180, 240, 360, and 480 post-dose. Glucosemeasurements were performed in whole blood using AlphaTRAK glucometers.If the glucose concentrations of two measurements differed by more than20 mg/dL a third value was recorded. The Area Under the Curve (AUC) forblood glucose was calculated via the trapezoid method and for theduration of the 24 post-dose hours.

SEQ ID NO: 6 or SEQ ID NO: 7 treated animals exhibited a comparable,acute decrease in blood glucose concentrations and reached lowest levelsapproximately 8 hours post dose (FIG. 10). Twenty four hours post-dosemean blood glucose concentrations of all treated animals were at orclose to baseline (FIG. 10). Single treatment of diabetic db/db micewith 30 μg/kg SEQ ID NO: 6 or 30 μg/kg SEQ ID NO: 7 resulted in astatistically significant reduction in the blood glucose AUC compared tothe Diabetic-Vehicle group (One-Way ANOVA and Tukey Test, FIG. 11, Table10).

2) Statistical Analyses

Data are depicted as means±SEM. Statistical analyses were performed withSigmaplot 12.5. A One-Way ANOVA and Tukey Test was used for pairwisemultiple comparisons of all groups (n=8/group). When the difference inthe mean values of the two groups was greater than 0.05 they wereconsidered statistically significantly different. Lean-Vehicle groupdata are depicted in the Figures serving as a reference dataset for thenon-obese state.

TABLE 10 Effects resulting from a single dose SEQ ID NO: 6 or SEQ ID NO:7 in fed, female db/db mice. Data are means ± SEM. One-Way ANOVA andTukey Test. n = 8/group. Diabetic- DIO-SEQ DIO-SEQ Example Vehicle IDNO: 6 ID NO: 7 Dose Once PBS Once 30 μg/kg Once 30 μg/kg Plasma glucosearea +705.52 ± 49.31 +430.70 ± 26.41 +461.54 ± 39.43 under the curve P <0.001 vs. P < 0.001 vs. (mmol/L in 24 h) Diabetic-VehicleDiabetic-Vehicle

Example 11: Acute and Chronic Effects of SEQ ID NO: 8, SEQ ID NO: 9 andSEQ ID NO: 10 after Subcutaneous Treatment on Blood Glucose and BodyWeight in Female Diet-Induced Obese (D10) C57BL/6NHsd Mice

Lean and diet-induced obese, 25-26 weeks old, female C57BL/6NHsd micewere treated twice daily with a s.c. injection of PBS, 30 μg/kg SEQ IDNO: 8, 30 μg/kg SEQ ID NO: 9, or 30 μg/kg SEQ ID NO: 10 for 28 days. Themorning dosing was initiated and completed between 06:00 and 07:30 AMand the afternoon dosing between 14:00 and 15:30 PM. The applied volumewas 5 ml/kg and the dose was adjusted to the most recent body massrecording of each individual.

1) Glucose Profile

Animals had unlimited access to water and feed during the experiment. Onday 1 of the dosing phase approximately 5 μL of blood were collected viatail clips at hour 0, prior to the first s.c. dose of PBS-vehicle, 30μg/kg SEQ ID NO: 8, 30 μg/kg SEQ ID NO: 9, or 30 μg/kg SEQ ID NO: 10 aswell as 1, 2, 3, 4, 6, and 24 hours post-dose. Animals received thesecond s.c. dose of PBS-vehicle, 30 μg/kg SEQ ID NO: 8, 30 μg/kg SEQ IDNO: 9, or 30 μg/kg SEQ ID NO: 10 8 hours after the first dose. The 24hour blood collection of the blood glucose profile was performed priorto dosing on day 2. Glucose measurements were performed in whole bloodand in duplicate or triplicate using Aviva glucometers.

30 μg/kg SEQ ID NO: 8, 30 μg/kg SEQ ID NO: 9, or 30 μg/kg, and SEQ IDNO: 10 mediated a comparable decrease in blood glucose concentrationsbelow concentrations observed in DIO control mice during the 24 hourblood glucose profile (FIG. 12).

2) Body Mass

Body mass was determined daily. In the dosing phase mice were treatedtwice daily with either a s.c. injection of PBS-vehicle, 30 μg/kg SEQ IDNO: 8, 30 μg/kg SEQ ID NO: 9, or 30 μg/kg SEQ ID NO: 10.

Chronic, twice daily treatment with SEQ ID NO: 8, SEQ ID NO: 9, or SEQID NO: 10 resulted in a body mass reduction within the 28 days of thedosing phase in contrast to animals of the DIO-vehicle group (FIG. 13).Chronic treatment of DIO animals with 30 μg/kg SEQ ID NO: 8, SEQ ID NO:9, or SEQ ID NO: 10 induced a statistically significant decrease in bodymass compared to the DIO-Vehicle group (One-Way ANOVA and Tukey Test,FIG. 14, Table 11).

3) Whole Body Fat Mass

Whole body fat mass was determined via Nuclear Magnetic Resonance (QNMR)once in the predosing phase and once on day 26 of the dosing phasedaily. In the dosing phase mice were treated twice daily with either as.c. injection of PBS-vehicle, 30 μg/kg SEQ ID NO: 8, 30 μg/kg SEQ IDNO: 9, or 30 μg/kg SEQ ID NO: 10. Chronic, twice daily treatment withSEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO resulted in a statisticallysignificantly reduction in whole body fat mass within the 28 dayscompared to the DIO-Vehicle group (One-Way ANOVA and Tukey Test, FIG.15, Table 11).

4) Terminal Liver Mass

On day 28 mice were euthanized 4 hours after the morning dose and liverswere collected.

Twice-daily, chronic treatment of DIO animals with 30 μg/kg SEQ ID NO:8, 30 μg/kg SEQ ID NO: 9, or 30 μg/kg SEQ ID NO: 10 resulted in astatistically significantly lower liver mass on day 28 of the dosingphase compared to DIO animals treated with vehicle (One-Way ANOVA andTukey Test, FIG. 16, Table 11).

5) Terminal Plasma Alanine Aminotransferase Concentration

On day 28 and 4 hours after the morning dose, blood was collected fromanesthetized, non-fasting mice by orbital bleeding.

Twice-daily, chronic treatment of DIO animals with 30 μg/kg SEQ ID NO:8, 30 μg/kg SEQ ID NO: 9, or 30 μg/kg SEQ ID NO: 10 resulted in astatistically significantly lower plasma alanine aminotransferaseconcentration on day 28 of the dosing phase compared to DIO animalstreated with vehicle suggesting improvement of liver function (One-WayANOVA and Tukey Test, FIG. 17, Table 11).

6) Terminal Plasma Total Cholesterol

On day 28 and 4 hours after the morning dose, blood was collected fromanesthetized, non-fasting mice by orbital bleeding.

Twice-daily, chronic treatment of DIO animals with 30 μg/kg SEQ ID NO:8, 30 μg/kg SEQ ID NO: 9, or 30 μg/kg SEQ ID NO: 10 resulted in astatistically significant reduction in plasma total cholesterolconcentrations compared to DIO animals treated with vehicle (One-WayANOVA and Tukey Test, FIG. 18, Table 11).

7) Terminal Plasma Insulin

On day 28 and 4 hours after the morning dose, blood was collected fromanesthetized, non-fasting mice by orbital bleeding.

Twice-daily, chronic treatment of DIO animals with 30 μg/kg SEQ ID NO:8, 30 μg/kg SEQ ID NO: 9, or 30 μg/kg SEQ ID NO: 10 resulted in astatistically significant reduction in plasma insulin concentrationscompared to DIO animals treated with vehicle hinting towards animprovement of high fat-diet induced insulin resistance (One-Way ANOVAand Tukey Test, FIG. 19, Table 11).

8) Statistical Analyses

Data are depicted as means±SEM. Statistical analyses were performed withSigmaplot 12.5. A One-Way ANOVA and Tukey Test was used for pairwisemultiple comparisons of all groups (as indicated n=7-8/group). When thedifference in the mean values of the two groups was greater than 0.05they were considered statistically significantly different. Lean-Vehiclegroup data are depicted in the Figures serving as a reference datasetfor the non-obese state.

TABLE 11 Effects resulting from a 28 days qd dosing study with SEQ IDNO: 8, ID NO: 9. or SEQ ID NO: 10 in fed, female diet-induced obese(DIO) C57BL/6 mice. Data are means ± SEM. One-Way ANOVA and Tukey Test,n = 8/group. DIO-Vehicle Example twice-daily DIO-SEQ ID NO: 8 DIO-SEQ IDNO: 9 DIO-SEQ ID NO: 10 Dose PBS twice-daily 30 μg/kg twice-daily 30μg/kg twice-daily 30 μg/kg Body mass  5.10 ± 0.49 −8.23 ± 0.62 −10.03 ±2.33 −7.14 ± 0.64 change (g) P < 0.001 vs. P < 0.001 vs. P < 0.001 vs.DIO-Vehicle DIO-Vehicle DIO-Vehicle Whole body fat  2.51 ± 0.65 −6.65 ±0.38  −5.29 ± 2.32 −6.48 ± 0.90 mass change (g) P < 0.001 vs. P < 0.001vs. P < 0.001 vs. DIO-Vehicle DIO-Vehicle DIO-Vehicle Terminal liver+1.80 ± 0.07 +1.08 ± 0.03  +1.11 ± 0.06 +1.07 ± 0.04 mass (g) P < 0.001vs. P < 0.001 vs. P < 0.001 vs. DIO-Vehicle DIO-Vehicle DIO-VehicleTerminal plasma 207.50 ± 14.98 +68.88 ± 15.05 +57.13 ± 7.33 +56.75 ±4.65  alanine P < 0.001 vs. P < 0.001 vs. P < 0.001 vs. aminotransferaseDIO-Vehicle DIO-Vehicle DIO-Vehicle (U/L) Terminal plasma +5.45 ± 0.15+4.36 ± 0.08  +3.97 ± 0.12 +4.28 ± 0.15 total cholesterol P < 0.001 vs.P < 0.001 vs. P < 0.001 vs. (mmol/L) DIO-Vehicle DIO-Vehicle DIO-VehicleTerminal plasma +145.08 ± 29.15  +39.70 ± 4.25  +38.95 ± 7.30 +35.81 ±2.79  insulin (U/L) P < 0.001 vs. P < 0.001 vs. P < 0.001 vs.DIO-Vehicle DIO-Vehicle DIO-Vehicle

Example 12: Acute Effects of SEQ ID NO: 13 after Subcutaneous Treatmenton Blood Glucose Concentrations in Non-Fasting, Female, Diabetic Db/DbMice

Female, 12 weeks old, healthy, lean BKS.Cg-(lean)/OlaHsd ordiabetes-prone, obese BKS.Cg-+Lepr^(db)/+Lepr^(db)/OlaHsd mice weretreated once with a s.c. injection of PBS or 30 μg/kg SEQ ID NO: 13. Thedosing was initiated and completed between 08:00 and 10:00 AM. Theapplied volume was 5 ml/kg and the dose was adjusted to the most recentbody mass recording of each individual. Diabetic db/db mice were HbA1c-and body mass-matched. The lean reference group was treated once with as.c. injection of PBS-vehicle.

1) Blood Glucose Profile

Animals had unlimited access to water and feed during the experiment. Onday 1 of the dosing phase approximately 5 μL of blood were collected viatail clips at minute −30 and 0 prior to any other inlife activities. Atminute 0 mice received a s.c. dose of either PBS-vehicle or 30 μg/kg SEQID NO: 13. Further blood samples were harvested at minute 15, 30, 60,90, 120, 150, 180, 240, 360, and 480 post-dose. Glucose measurementswere performed in whole blood using AlphaTRAK glucometers. If theglucose concentrations of two measurements differed by more than 20mg/dL a third value was recorded. The Area Under the Curve (AUC) forblood glucose was calculated via the trapezoid method and for theduration of the 24 post-dose hours.

SEQ ID NO: 13 treated animals exhibited an acute decrease in bloodglucose concentrations and reached lowest levels approximately 8 hourspost dose (FIG. 20). Twenty four hours post-dose mean blood glucoseconcentrations of SEQ ID NO: 13 treated animals were close to baseline(FIG. 20). Single treatment of diabetic db/db mice with 30 μg/kg SEQ IDNO: 13 resulted in a statistically significant reduction in the bloodglucose AUC compared to the Diabetic-Vehicle group (One-Way ANOVA andTukey Test, FIG. 21, Table 12).

2) Statistical Analyses

Data are depicted as means±SEM. Statistical analyses were performed withSigmaplot 12.5. A One-Way ANOVA and Tukey Test was used for pairwisemultiple comparisons of all groups (n=8/group). When the difference inthe mean values of the two groups was greater than 0.05 they wereconsidered statistically significantly different. Lean-Vehicle groupdata are depicted in the Figures serving as a reference dataset for thenon-obese state.

TABLE 12 Effects resulting from a single dose SEQ ID NO: 13 in fed,female db/db mice. Data are means ± SEM. One-Way ANOVA and Tukey Test. n= 8/group. Example Diabetic-Vehicle DIO-SEQ ID NO: 13 Dose Once PBS Once30 μg/kg Plasma glucose area +646.38 ± 45.04 +458.34 ± 27.11 under thecurve P < 0.001 vs. (mmol/L in 24 h) Diabetic-Vehicle

TABLE 13 Sequences SEQ. ID sequence  1H-A-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-K-E-F-I-A-W-L-V-K-G-R- NH2  2H-A-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-K(gGlu-Palm)-E-F-I-A-W-L-V-R-G-R-G-OH  3H-S-Q-G-T-F-T-S-D-Y-S-K-Y-L-D-S-R-R-A-Q-D-F-V-Q-W-L-M-N-T- OH  4H-G-E-G-T-F-T-S-D-L-S-K-Q-M-E-E-E-A-V-R-L-F-I-E-W-L-K-N-G-G-P-S-S-G-A-P-P-P-S-NH2  5Y-A-E-G-T-F-I-S-D-Y-S-I-A-M-D-K-I-H-Q-Q-D-F-V-N-W-L-L-A-Q-K-G-K-K-N-D-W-K-H-N-I-T-Q-OH  6H-Aib-Q-G-T-F-T-S-D-L-S-K-L-K[gGlu-gGlu-Palm]-E-K-Q-R-Q-Aib-E-F-I-E-W-L-K-A-G-G-P-P-S-Aib-K-P-P-P-K-NH2  7H-Aib-Q-G-T-F-T-S-D-L-S-K-L-K[gGlu-gGlu-Palm]-E-K-Q-R-Q-Aib-E-F-I-E-W-L-K-A-dAla-G-P-P-S-Aib-K-P-P-P-K-NH2  8H-Aib-Q-G-T-F-T-S-D-L-S-K-L-K[gGlu-gGlu-Palm]-E-E-Q-R-Q-Aib-E-F-I-E-W-L-K-A-G-G-H-P-S-Aib-K-P-P-P-K-NH2  9H-Aib-Q-G-T-F-T-S-D-L-S-K-L-K[gGlu-gGlu-Palm]-E-E-Q-R-Q-Aib-E-F-I-E-W-L-K-A-G-G-P-P-S-Aib-K-P-P-P-K-NH2 10H-Aib-Q-G-T-F-T-S-D-L-S-K-L-K[gGlu-gGlu-Palm]-E-E-Q-R-Q-Aib-E-F-I-E-W-L-K-A-dAla-G-P-P-S-Aib-K-P-P-P-K-NH2 11H-Aib-Q-G-T-F-T-S-D-L-S-K-L-K[gGlu-gGlu-Palm]-E-E-Q-R-Q-Aib-E-F-I-E-W-L-K-A-dAla-G-H-P-S-Aib-K-P-P-P-K-NH2 12H-Aib-Q-G-T-F-T-S-D-L-S-K-L-K[gGlu-gGlu-Palm]-E-K-Q-R-Q-Aib-E-F-I-E-W-L-K-A-G-G-H-P-S-Aib-K-P-P-P-K-NH2 13H-Aib-Q-G-T-F-T-S-D-L-S-K-L-K[gGlu-gGlu-Palm]-E-K-Q-R-Q-Aib-E-F-I-E-W-L-K-A-dAla-G-H-P-S-Aib-K-P-P-P-K-NH2 14H-Aib-Q-G-T-F-T-S-D-L-S-K-L-K[gGlu-Palm]-E-K-Q-R-Q-Aib-E-F-I-E-W-L-K-A-G-G-P-P-S-Aib-K-P-P-P-K-NH2 15H-Aib-Q-G-T-F-T-S-D-L-S-K-L-K[gGlu-Stea]-E-K-Q-R-Q-Aib-E-F-I-E-W-L-K-A-G-G-P-P-S-Aib-K-P-P-P-K-NH2 16H-Aib-Q-G-T-F-T-S-D-L-S-K-L-K[gGlu-gGlu-AEEA-Palm]-E-K-Q-R-Q-Aib-E-F-I-E-W-L-K-A-G-G-H-P-S-Aib-K-P-P-P-K-NH2 17H-Aib-Q-G-T-F-T-S-D-L-S-K-L-K[gGlu-gGlu-AEEA-Palm]-E-K-Q-R-Q-Aib-E-F-I-E-W-L-K-A-dAla-G-H-P-S-Aib-K-P-P-P-K-NH2 18H-Aib-Q-G-T-F-T-S-D-L-S-K-L-K[gGlu-gGlu-AEEA-Palm]-E-E-Q-R-Q-Aib-E-F-I-E-W-L-K-A-G-G-H-P-S-Aib-K-P-P-P-K-NH2 19H-Aib-Q-G-T-F-T-S-D-L-S-K-L-K[gGlu-gGlu-AEEA-Palm]-E-E-Q-R-Q-Aib-E-F-I-E-W-L-K-A-dAla-G-H-P-S-Aib-K-P-P-P-K-NH2 20H-Aib-Q-G-T-F-T-S-D-L-S-K-L-K[gGlu-AEEA-gAAA-Palm]-E-K-Q-R-Q-Aib-E-F-I-E-W-L-K-A-G-G-H-P-S-Aib-K-P-P-P-K-NH2 21H-Aib-Q-G-T-F-T-S-D-L-S-K-L-K[gGlu-AEEA-gAAA-Palm]-E-K-Q-R-Q-Aib-E-F-I-E-W-L-K-A-dAla-G-H-P-S-Aib-K-P-P-P-K-NH2 22H-Aib-Q-G-T-F-T-S-D-L-S-K-L-K[gGlu-AEEA-gAAA-Palm]-E-E-Q-R-Q-Aib-E-F-I-E-W-L-K-A-G-G-H-P-S-Aib-K-P-P-P-K-NH2 23H-Aib-Q-G-T-F-T-S-D-L-S-K-L-K[gGlu-AEEA-gAAA-Palm]-E-E-Q-R-Q-Aib-E-F-I-E-W-L-K-A-dAla-G-H-P-S-Aib-K-P-P-P-K-NH2

1. A compound of the formula I: IH₂N-His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Leu-X14-Glu-X16-Gln-Arg-Gln-Aib-Glu-Phe-Ile-Glu-Trp-Leu-Lys-Ala-X29-Gly-X31-Pro-Ser-Aib-Lys- Pro-Pro-Pro-Lys-R¹

wherein: X14 is an amino acid residue with a functionalized —NH₂ sidechain group, selected from the group consisting of Lys, Orn, Dab, andDap, wherein the —NH₂ side chain group is functionalized by —Z—C(O)—R⁵,wherein Z is a linker in all stereoisomeric forms and R⁵ is a moietycomprising up to 50 carbon atoms and heteroatoms selected from N and O;X16 is an amino acid residue selected from Glu and Lys; X29 is an aminoacid residue selected from D-Ala and Gly; X31 is an amino acid residueselected from His and Pro; and R¹ is NH₂ or OH; or a salt or solvatethereof.
 2. The compound of claim 1, wherein R¹ is NH₂, or a salt orsolvate thereof.
 3. The compound of claim 1, or a salt or solvatethereof, which has a relative activity of at least 1% compared to thatof natural glucagon at the glucagon receptor.
 4. The compound of claim1, or a salt or solvate thereof, which exhibits a relative activity ofat least 10% compared to that of GLP-1(7-36)-amide at the GLP-1receptor.
 5. The compound of claim 1, or a salt or solvate thereof,which exhibits a relative activity of at least 2% compared to that ofGIP at the GIP receptor.
 6. The compound of claim 1, wherein X14 is Lys,wherein the —NH₂ side chain group is functionalized with a group—Z—C(O)R⁵, wherein: Z is a group selected from gGlu, gGlu-gGlu,gGlu-AEEAc-gAAA-, gGlu-gGlu-AEEAc, AEEAc-AEEAc-gGlu andAEEAc-AEEAc-AEEAc, and R⁵ is a group selected from pentadecanyl andheptadecanyl; or a salt or solvate thereof.
 7. The compound of claim 1,wherein X14 is Lys, wherein the —NH₂ side chain group is functionalizedwith a group —Z—C(O)R⁵, wherein: Z is a group selected from gGlu,gGlu-gGlu, gGlu-AEEAc-gAAA- and gGlu-gGlu-AEEAc; and R⁵ is a groupselected from pentadecanyl and heptadecanyl; or a salt or solvatethereof.
 8. The compound of claim 1, wherein: X14 is Lys, wherein the—NH₂ side chain group is functionalized by a group selected from(S)-4-Carboxy-4-hexadecanoylamino-butyryl-,(S)-4-Carboxy-4-octadecanoylamino-butyryl-,(S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-,(2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-hexadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl,(2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-octadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl,and[2-(2-{2-[2-(2-{2-[2-(2-Octadecanoylamino-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl-,and R¹ is NH₂; or a salt or solvate thereof.
 9. The compound of claim 1,wherein: X14 is Lys, wherein the —NH₂ side chain group is functionalizedby a group selected from (S)-4-Carboxy-4-hexadecanoylamino-butyryl-,(S)-4-Carboxy-4-octadecanoylamino-butyryl-,(S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-,(2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-hexadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl,and(2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-octadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl,and R¹ is NH₂; or a salt or solvate thereof.
 10. The compound of claim1, wherein: X14 is Lys, wherein the —NH₂ side chain group isfunctionalized by(S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-;X16 is an amino acid residue selected from Glu and Lys; X29 is an aminoacid residue selected from D-Ala and Gly; X31 is an amino acid residueselected from His and Pro; and R¹ is NH₂; or a salt or solvate thereof.11. The compound of claim 1, wherein: X14 is Lys, wherein the —NH₂ sidechain group is functionalized by a group selected from(S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-and (S)-4-Carboxy-4-octadecanoylamino-butyryl-; X16 is an amino acidresidue selected from Glu and Lys; X29 is Gly; X31 is an amino acidresidue selected from His and Pro; and R¹ is NH₂; or a salt or solvatethereof.
 12. The compound of claim 1, wherein: X14 is Lys, wherein the—NH₂ side chain group is functionalized by a group selected from(S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-and (S)-4-Carboxy-4-octadecanoylamino-butyryl-; X16 is Lys; X29 is anamino acid residue selected from D-Ala and Gly; X31 is an amino acidresidue selected from His and Pro; and R¹ is NH₂; or a salt or solvatethereof.
 13. The compound of claim 1, wherein: X14 is Lys, wherein the—NH₂ side chain group is functionalized by a group selected from(S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-and (S)-4-Carboxy-4-octadecanoylamino-butyryl-; X16 is an amino acidresidue selected from Glu and Lys; X29 is an amino acid residue selectedfrom D-Ala and Gly; X31 is Pro; and R¹ is NH₂; or a salt or solvatethereof.
 14. The compound of claim 1, selected from the compounds of SEQID NOs: 6-23, or salts or solvates thereof.
 15. The compound of claim 1,wherein the compound is the compound of SEQ ID NO: 6, or a salt orsolvate thereof.
 16. The compound of claim 1, wherein the compound isthe compound of SEQ ID NO: 9, or a salt or solvate thereof.
 17. Thecompound of claim 1, wherein the compound is the compound of SEQ ID NO:11, or a salt or solvate thereof.
 18. A pharmaceutical compositioncomprising the compound of claim 1, or a salt or solvate thereof.
 19. Apharmaceutical composition comprising the compound of claim 1, or apharmaceutically acceptable salt or solvate thereof, which is present asan active agent together with at least one pharmaceutically acceptablecarrier.
 20. The pharmaceutical composition of claim 18, furthercomprising at least one additional therapeutically active agent.
 21. Thepharmaceutical composition of claim 20, wherein the at least oneadditional therapeutically active agent is selected from the groupconsisting of: insulin and insulin derivatives; GLP-1, GLP-1 analoguesand GLP-1 receptor agonists; DPP-4 inhibitors; SGLT2 inhibitors; dualSGLT2/SGLT1 inhibitors; biguanides, thiazolidinediones, dual PPARagonists, sulfonylureas, meglitinides, alpha-glucosidase inhibitors,amylin and amylin analogues; GPR119 agonists, GPR40 agonists, GPR120agonists, GPR142 agonists, systemic or low-absorbable TGR5 agonists;bromocriptine mesylate, inhibitors of 11-beta-HSD, activators ofglucokinase, inhibitors of DGAT, inhibitors of proteintyrosine-phosphatase 1, inhibitors of glucose-6-phosphatase, inhibitorsof fructose-1,6-bisphosphatase, inhibitors of glycogen phosphorylase,inhibitors of phosphoenol pyruvate carboxykinase, inhibitors of glycogensynthase kinase, inhibitors of pyruvate dehydrokinase, alpha2-antagonists, CCR-2 antagonists, SGLT-1 inhibitors, modulators ofglucose transporter-4, somatostatin receptor 3 agonists; lipid loweringagents; active substances for the treatment of obesity; gastrointestinalpeptides; lipase inhibitors, angiogenesis inhibitors, H3 antagonists,AgRP inhibitors, triple monoamine uptake inhibitors, MetAP2 inhibitors,nasal formulation of the calcium channel blocker diltiazem, antisensemolecules against production of fibroblast growth factor receptor 4,prohibitin targeting peptide-1; and drugs for influencing high bloodpressure, chronic heart failure or atherosclerosis.
 22. A method for thetreatment of glucose intolerance, insulin resistance, pre-diabetes,increased fasting glucose, hyperglycemia, type 2 diabetes, hypertension,dyslipidemia, arteriosclerosis, coronary heart disease, peripheralartery disease, stroke or any combination of these individual diseasecomponents in a patient, the method comprising administering to thepatient the compound of claim 1, or a salt or solvate thereof.
 23. Amethod for control of appetite, feeding and calorie intake, increase ofenergy expenditure, prevention of weight gain, promotion of weight loss,reduction of excess body weight, obesity, and morbid obesity, in apatient, the method comprising administering to the patient the compoundof claim 1, or a salt or solvate thereof.
 24. A method for the treatmentor prevention of hepatosteatosis in a patient, the method comprisingadministering to the patient the compound of claim 1, or a salt orsolvate thereof.
 25. The method of claim 22 for the treatment orprevention of hyperglycemia, diabetes, or obesity.
 26. The method ofclaim 25 for the simultaneous treatment of diabetes and obesity.
 27. Themethod of claim 25 for the treatment of type 2 diabetes.
 28. The methodof claim 25 for the treatment of obesity.
 29. The method of claim 28 forreducing body weight of a patient.
 30. A method for reducing theintestinal passage, increasing the gastric content and/or reducing thefood intake of a patient, the method comprising administering to thepatient the compound of claim 1, or a salt or solvate thereof.
 31. Amethod for reducing blood glucose levels and/or reducing HbA1c levels ofa patient, the method comprising administering to the patient thecompound of claim 1, or a salt or solvate thereof.
 32. A method fortreating hyperglycemia, type 2 diabetes or obesity in a patient, themethod comprising administering to the patient an effective amount of atleast one compound of claim 1, or a salt or solvate thereof, and aneffective amount of at least one other compound useful for treatingdiabetes, obesity, dyslipidemia, or high blood pressure.
 33. The methodof claim 32, wherein the effective amount of the at least one compound,or the salt or solvate thereof, and the additional active ingredient areadministered to the patient simultaneously.
 34. The method of claim 32,wherein the effective amount of the at least one compound, or the saltor solvate thereof, and the additional active ingredient areadministered to the patient sequentially.